*Article* **Quantitative Anatomical Characteristics of Virgin Cork in** *Quercus variabilis* **Grown in Korea**

**Denni Prasetia <sup>1</sup> , Byantara Darsan Purusatama <sup>2</sup> , Jong-Ho Kim <sup>1</sup> , Go-Un Yang <sup>1</sup> , Jae-Hyuk Jang <sup>3</sup> , Se-Yeong Park <sup>1</sup> , Seung-Hwan Lee <sup>1</sup> and Nam-Hun Kim 1,\***


**Abstract:** The quantitative anatomical characteristics of *Quercus variabilis* virgin cork grown in Korea were observed by scanning electron microscopy and compared with *Quercus suber* reproduction cork from Portugal to obtain basic data for further utilization of domestic cork resources in Korean cork industries. *Q. variabilis* virgin cork showed a smaller growth ring width and higher latecork percentage than *Q. suber* reproduction cork. *Q. variabilis* showed a smaller proportion of cork cells and a higher proportion of lenticular channels than *Q. suber*, whereas sclereid and dark-brown zones were found only in *Q. variabilis.* The frequency of pentagonal cork cells in the transverse and radial sections was higher in the cork of *Q. suber* than in *Q. variabilis.* In the tangential section, *Q. variabilis* displayed a lower frequency of heptagonal cells and a higher frequency of pentagonal cells than *Q. suber. Q. variabilis* cork had a smaller cell width, lumen diameter, cell wall thickness, prism base edge and area, total cell volume, and solid volume of the cell wall than *Q. suber cork.* The fractional solid volume and number of cells per cm<sup>3</sup> were higher in *Q. variabilis* than *Q. suber*.

**Keywords:** dark-brown zone; lenticular filling tissue; quantitative anatomical characteristics; *Quercus suber*; *Quercus variabilis*; reproduction cork; scanning electron microscopy; sclereid; virgin cork

### **1. Introduction**

Cork is a non-timber forest product and a part of the periderm in the bark system that surrounds the stems, branches, and roots of dicotyledonous plants [1]. Cork has high economic value owing to its remarkable properties, including its impermeability to liquids and gases, its excellent thermal and sound insulation, shock absorption [2,3], and a high coefficient of friction and resistance to microbial activity [4]. Therefore, cork is widely used as a renewable and sustainable raw material in industry, including in wine stoppers [1], floors and ceilings [5], food product packaging [6], insulation for energy absorption [7], and the surfacing of walking areas [8].

There are three types of cork in the process of cork production: virgin cork, second cork, and reproduction cork. Virgin cork is found in the first periderm [9], while second cork is produced by the regenerated phellogen after removing virgin cork. Successive cork layers are called reproduction cork and are harvested from trees at nine-year intervals [4]. Virgin and second corks are generally used as triturations for agglomerate production because of the uneven structure of the cork tissue. In contrast, reproduction cork is the most essential material in the cork industry owing to its structure for producing solid cork products, such as wine stoppers [1].

The primary resource of cork in the global industry is that obtained from *Q. suber*, a species of plant that is widely distributed in the Western Mediterranean; Southwestern Europe, including Portugal, Spain, southern France, and Italy; and in North Africa, such as Morocco, Algeria, and Tunisia [10]. *Q. variabilis* is another species that contains a large

**Citation:** Prasetia, D.; Purusatama, B.D.; Kim, J.-H.; Yang, G.-U.; Jang, J.-H.; Park, S.-Y.; Lee, S.-H.; Kim, N.-H. Quantitative Anatomical Characteristics of Virgin Cork in *Quercus variabilis* Grown in Korea. *Forests* **2022**, *13*, 1711. https:// doi.org/10.3390/f13101711

Academic Editors: Vicelina Sousa, Helena Pereira, Teresa Quilhó and Isabel Miranda

Received: 13 September 2022 Accepted: 15 October 2022 Published: 17 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

amount of cork in its bark periderm, widely found in Eastern Asia, including China, Korea, and Japan [11]. The cork of *Q. variabilis* has been cultivated and exploited in China for a limited scale of cork production [12].

The anatomical characteristics of cork, including growth ring characteristics, cork element composition, and cork cell structure, are the basis of many properties of the materials, such as very low permeability, hydrophobic behavior, biological inertia, high elasticity in compression, and dimensional recovery [1,13–15]. Many studies have reported on the quantitative aspects of the anatomical characteristics of cork in *Q. variabilis* grown in China. According to Yafang et al. [16], the three-dimensional structure of virgin cork cells in *Q. variabilis* was a prism, with the cork frequently showing a hexagonal shape in the transverse, radial, and tangential sections (53.6%, 52.5%, and 52.3% of cells, respectively). These authors also mentioned that cork cells showed a pentagonal shape in the transverse, radial, and tangential sections (22.6%, 23.4%, and 25.90%, respectively), as well as a heptagonal shape in the transverse, radial, and tangential sections (19.5%, 14.9%, and 14.3%, respectively). Additionally, the prism height was found to decrease from earlycork to latecork cells, whereas the radial cell wall thickness increased from earlycork to latecork cells. The prism base edge of the earlycork cells was 8.0–14.0 µm, with an average base area of 200–500 µm<sup>2</sup> . Miranda et al. [12] reported that *Q. variabilis* reproduction cork frequently showed a hexagonal shape in the tangential section, which accounted for approximately 60.0% of cork cells. In the non-tangential sections, 39.5% of the cork cells were pentagonal, while 37.1% were hexagonal. The earlycork cells of *Q. variabilis* showed a higher prism height and total cell volume than latecork cells, while the radial cell wall thickness and solid volume fraction increased from earlycork to latecork cells. Ferreira et al. [13] reported that 40.5% and 41.2% of the cells in the tangential section of the virgin and reproduction corks in *Q. variabilis* were hexagonal, while 31.3% and 32.1% were pentagonal. In the non-tangential sections, virgin cork dominantly showed a pentagonal shape (44.4% of cells), while reproduction cork commonly showed a hexagonal shape (46.9% of cells). The prism height, total cell volume, and solid volume fraction in virgin cork were smaller than in reproduction cork; however, the prism base edge and average base area in virgin cork were larger than in reproduction cork. Li et al. [17] reported that the virgin and reproduction corks of *Q. variabilis* displayed lenticular channels and sclereids surrounded by dark and hard layers. In addition, lenticular channels and sclereids surrounded by dark and hard layers of the reproduction cork were less frequent than those of the virgin cork.

At present, Kim [18] investigated the quantitative anatomical characteristics of *Q. variabilis* reproduction cork grown in Korea and reported that *Q. variabilis* reproduction cork consisted of 87.2% cork cells, 9.0% lenticels, 0.8% sclereids, and 3.0% dark-brown zones. The growth ring width of *Q. variabilis* was found to be narrower than *Q. suber* reproduction cork (0.82 mm and 2.06 mm, respectively). Earlycork cells showed a higher prism height than latecork cells, whereas the prism edge length and cell wall thickness were greater in latecork than in earlycork cells.

In Korea, *Q. variabilis* wood has been used historically as a raw material for timber, firewood, charcoal [19–21], musical instruments, and fuel [22]. *Q. variabilis* also has a large amount of cork in its bark, which can be used as a sustainable resource in the cork industry [13]; nevertheless, no reproduction cork from *Q. variabilis* is available for industrial use in Korea. Therefore, *Q. suber* reproduction cork is largely imported from Portugal.

In Korean cork industry, the main raw material for various products (e.g., wine stoppers, insulation boards, and surfacing products for pavements and sidewalls) is *Q. suber* reproduction cork imported from Portugal; however, with an increasing demand for cork products, there is a need to identify alternative cork resources from domestic oak species to ensure the success of the Korean cork industry. In Korea, *Q. variabilis* is widely distributed, and virgin cork can be easily obtained from the trees; however, there remains a lack of information regarding the quality of cork resources produced by this species. Thus, to gain insights into cork quality and effectively utilize the cork of *Q. variabilis* as sustainable raw material for various products, the quantitative anatomical characteristics of *Q. variabilis*

virgin cork grown in Korea were investigated and compared with those of commercial cork (*Q. suber* reproduction cork from Portugal). *bilis* virgin cork grown in Korea were investigated and compared with those of commercial cork (*Q. suber* reproduction cork from Portugal).

the cork industry [**Error! Reference source not found.**]; nevertheless, no reproduction cork from *Q. variabilis* is available for industrial use in Korea. Therefore, *Q. suber* repro-

In Korean cork industry, the main raw material for various products (e.g., wine stoppers, insulation boards, and surfacing products for pavements and sidewalls) is *Q. suber* reproduction cork imported from Portugal; however, with an increasing demand for cork products, there is a need to identify alternative cork resources from domestic oak species to ensure the success of the Korean cork industry. In Korea, *Q. variabilis* is widely distributed, and virgin cork can be easily obtained from the trees; however, there remains a lack of information regarding the quality of cork resources produced by this species. Thus, to gain insights into cork quality and effectively utilize the cork of *Q. variabilis* as sustainable raw material for various products, the quantitative anatomical characteristics of *Q. varia-*

*Forests* **2022**, *13*, x FOR PEER REVIEW 3 of 15

duction cork is largely imported from Portugal.

#### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. Materials 2.1. Materials*

*Quercus variabilis* virgin cork was collected from three trees at breast height in the research forest of Kangwon National University, Chuncheon, Korea (37◦770 N, 127◦810 E). Two planks of *Q. suber* reproduction cork from Castelo Branco cork forest of Amorim Group (Mozelos, Portugal) were provided by FC Korea Land Co., Ltd. (Seoul, Korea). Photographs of *Q. variabilis* virgin cork and *Q. suber* reproduction cork are presented in Figure 1, and basic information on the cork samples is presented in Table 1. *Quercus variabilis* virgin cork was collected from three trees at breast height in the research forest of Kangwon National University, Chuncheon, Korea (37°77′ N, 127°81′ E). Two planks of *Q. suber* reproduction cork from Castelo Branco cork forest of Amorim Group (Mozelos, Portugal) were provided by FC Korea Land Co., Ltd. (Seoul, Korea). Photographs of *Q. variabilis* virgin cork and *Q. suber* reproduction cork are presented in Figure 1, and basic information on the cork samples is presented in Table 1.

**Figure 1.** *Q. variabilis* virgin cork (**A**) and *Q. suber* reproduction cork (**B**). Black scale bar represents 20 mm. **Figure 1.** *Q. variabilis* virgin cork (**A**) and *Q. suber* reproduction cork (**B**). Black scale bar represents 20 mm.

**Table 1.** Basic information of the sample cork. **Table 1.** Basic information of the sample cork.


#### gitudinal) mm3 were prepared, and the transverse section was trimmed using a sliding *2.2. Measurement of Growth Ring Characteristics*

microtome (MSL-H; Nippon Optical Works, Nagano, Japan). The growth ring characteristics, such as quantity, ring width, and percentage of latecork, in both species were measured in the transverse section of three samples from each species. The growth ring number and width were observed using a measuring microscope (MM-40; Nikon, Tokyo, Japan) connected to an image analysis system (IMT i-solution lite, version 9.1; Burnaby, British Columbia, Canada). For latecork percentage, the cork samples with dimensions of 10 (radial) × 10 (tangential) × 10 (longitudinal) mm3 were coated with gold using a sputter coater Samples of both species with dimensions of 20–40 (radial) × 20 (tangential) × 20 (longitudinal) mm<sup>3</sup> were prepared, and the transverse section was trimmed using a sliding microtome (MSL-H; Nippon Optical Works, Nagano, Japan). The growth ring characteristics, such as quantity, ring width, and percentage of latecork, in both species were measured in the transverse section of three samples from each species. The growth ring number and width were observed using a measuring microscope (MM-40; Nikon, Tokyo, Japan) connected to an image analysis system (IMT i-solution lite, version 9.1; Burnaby, British Columbia, Canada). For latecork percentage, the cork samples with dimensions of 10 (radial) <sup>×</sup> 10 (tangential) <sup>×</sup> 10 (longitudinal) mm<sup>3</sup> were coated with gold using a sputter coater (Cressington sputter coater 108; Watford, UK) and observed using a scanning electron microscope (SEM) (JSM-5510, 15 kV; Tokyo, Japan).

Samples of both species with dimensions of 20–40 (radial) × 20 (tangential) × 20 (lon-

#### *2.3. Cellular Structure Observations*

Samples with dimensions of 20 (radial) <sup>×</sup> 20 (tangential) <sup>×</sup> 20 (longitudinal) mm<sup>3</sup> were prepared, and the transverse, radial, and tangential sections were trimmed using a sliding microtome (MSL-H; Nippon Optical Works, Nagano, Japan). The cork tissue, such as cork cells, lenticular channel, sclereid, and dark-brown zone, were determined as the ratio of the area of each element to the total area of 400 mm<sup>2</sup> , and the measurements were performed using 20 samples from each species. The images of the three sections were

captured using a mobile phone and recorded in digital format of 3024 × 3024 pixels with a resolution of 72 dpi and 24-bits depth (Samsung Note 20, 12MP with F1.8; Suwon-si, Gyeonggi-do, Korea). The images were analyzed using ImageJ (version Java 1.80\_172, 64 bits; Bethesda, MD, USA).

#### *2.4. Cork cell Dimension*

Cork samples with dimensions of 10 (radial) × 10 (tangential) × 10 (longitudinal) mm<sup>3</sup> were used to observe cork cell dimensions. The cork samples were coated with gold using a sputter coater (Cressington sputter coater 108; Watford, UK) and observed using SEM (JSM-5510, 15 kV; Tokyo, Japan).

The number of edges and the two- and three-dimensional characteristics were detected from 400 cork cells in both species.

The frequency of the edge number in a cork cell (*f i*) was measured in the transverse, radial, and tangential sections and calculated using Equation (1). Then, the dispersion (*µ*2) of the function in relation to the mean of cell shape (*im*) was calculated using Equation (2) [4]. The dispersion value presented homogeneity of the cell shape in each section of both species. If all cells show one type of cell shape, the dispersion value would be 0 [12].

$$f i = \left( \text{Ni} / \sum \text{Ni} \right) \times 100 \text{ (\%)} \tag{1}$$

$$\mu\_2 = \sum (i - i\_m)^2 \times fi \, (\%) \, \tag{2}$$

where *Ni* represents the number of cells with *i* is edges, and ∑ *Ni* is the total number of cells.

The two-dimensional characteristics of earlycork and latecork cells, such as radial width or prism height, radial lumen diameter, radial wall thickness, tangential width, tangential lumen diameter, and tangential wall thickness, were measured in the transverse section. In addition, the radial and tangential wall thicknesses of the earlycork and latecork cells were calculated using Equation (3) [12], as follows:

$$\text{Cell wall thickness} = (\text{cell dimension} - \text{lumen dimension})/2 \,\text{(\mu m)}\tag{3}$$

A three-dimensional illustration of a cork cell as a hexagonal prism is shown in Figure 2. The three-dimensional characteristics of earlycork and latecork cells, including radial width or prism height, total cell volume, solid volume, fractional solid volume of cell walls, and number of cork cells per cm<sup>3</sup> , were measured in the transverse section. Additionally, the prism base edge and base area of earlycork cells were measured in the tangential section. *Forests* **2022**, *13*, x FOR PEER REVIEW 5 of 15

**Figure 2.** Three-dimensional illustration of a cork cell as a hexagonal prism (modified from Miranda et al. [**Error! Reference source not found.**]). **Figure 2.** Three-dimensional illustration of a cork cell as a hexagonal prism (modified from Miranda et al. [12]).

The total volume (*V*), solid volume (*Vs*), and lumen volume (*Vo*) of earlycork and latecork cells were calculated according to Pereira [1], as shown in Equation (4). The fractional

× (ℎ−) (μm3)

Statistical differences in the quantitative anatomical characteristics between species were analyzed using one-way analysis of variance (ANOVA), followed by post hoc Tukey's honestly significant difference (HSD) test (*p* ≤ 0.05) (SPSS, version 24; IBM Corp.,

The growth ring widths in *Q. variabilis* virgin cork and *Q. suber* reproduction cork are shown in Table 2. The growth ring width was 0.54 mm for *Q. variabilis* cork and 3.07 mm for *Q. suber* cork. A significant difference was observed in terms of the width of the growth

Note: Numbers in parentheses are standard deviations (SD). The listing of the same superscript lowercase letters beside the mean values in the same row denotes nonsignificant outcomes at the

**Table 2.** Growth ring widths of *Q. variabilis* virgin cork and *Q. suber* reproduction cork.

*Q. variabilis* **(mm)** *Q. suber* **(mm)**  Tree 1 Tree 2 Tree 3 Average Plank 1 Plank 2 Average 0.58 (0.24) 0.61 (0.2) 0.43 (0.22) 0.54 a (0.10) 3.08 (0.54) 3.05 (0.26) 3.07 b (0.02)

5% significance level for comparisons between species.

୴ୣ୰ୟୣ ୡ୭୰୩ ୡୣ୪୪ ୴୭୪୳୫ୣ ୧୬ ୣୟ୰୪୷ୡ୭୰୩ ୟ୬ୢ ୪ୟ୲ୣୡ୭୰୩ ୡୣ୪୪ୱ (6)

(5)

× (ℎ−) (μm3)

where is the base edge, ℎ is the prism height, and is the wall thickness. The number of cork cells per cm3 (N) was calculated using Equation (6).

<sup>ଶ</sup> ቀ − √ଷ ቁ ଶ

N = େ୭୰୩ ୴୭୪୳୫ୣ ୭ ଵ ୡ୫య

solid volume (*Vsf)* was calculated using Equation (5):

*<sup>V</sup>* = 3ටଷ

*V0* = 3ට<sup>ଷ</sup>

*Vs* = 3ටଷ

*Vsf* = ೞ 

*2.5. Statistical Analysis* 

New York, NY, USA).

**3. Results and Discussion**  *3.1. Growth Ring Characteristics* 

ring between the two species.

<sup>ଶ</sup> ቀ − √ଷ ቁ ଶ

<sup>ଶ</sup> <sup>ଶ</sup> × ℎ <sup>−</sup>3ටଷ

× 100 (%)

The total volume (*V*), solid volume (*Vs*), and lumen volume (*Vo*) of earlycork and latecork cells were calculated according to Pereira [1], as shown in Equation (4). The fractional solid volume (*Vsf*) was calculated using Equation (5):

$$V = 3\sqrt{\frac{3}{2}}i^2 \times h \,\left(\mu\text{m}^3\right) \tag{4}$$

$$\begin{array}{l} V\_0 = 3\sqrt{\frac{3}{2}} \left( i - \frac{e}{\sqrt{3}} \right)^2 \times (h - e) \text{ (\mu m $^3$ )}\\ V\_s = 3\sqrt{\frac{3}{2}}i^2 \times h - 3\sqrt{\frac{3}{2}} \left( i - \frac{e}{\sqrt{3}} \right)^2 \times (h - e) \text{ (\mu m $^3$ )}\\ V\_{sf} = \frac{V\_s}{V} \times 100 \text{ (\%)} \end{array} \tag{5}$$

where *i* is the base edge, *h* is the prism height, and *e* is the wall thickness.

The number of cork cells per cm<sup>3</sup> (N) was calculated using Equation (6).

$$\text{N} = \frac{\text{Cork volume of 1 cm}^3}{\text{Average rock cell volume in earlyrock and latest cells}} \tag{6}$$

#### *2.5. Statistical Analysis*

Statistical differences in the quantitative anatomical characteristics between species were analyzed using one-way analysis of variance (ANOVA), followed by post hoc Tukey's honestly significant difference (HSD) test (*p* ≤ 0.05) (SPSS, version 24; IBM Corp., New York, NY, USA).

#### **3. Results and Discussion**

#### *3.1. Growth Ring Characteristics*

The growth ring widths in *Q. variabilis* virgin cork and *Q. suber* reproduction cork are shown in Table 2. The growth ring width was 0.54 mm for *Q. variabilis* cork and 3.07 mm for *Q. suber* cork. A significant difference was observed in terms of the width of the growth ring between the two species.



Note: Numbers in parentheses are standard deviations (SD). The listing of the same superscript lowercase letters beside the mean values in the same row denotes nonsignificant outcomes at the 5% significance level for comparisons between species.

The radial variation in the growth ring width from the cork back to the phellogen is shown in Figure 3. *Q. variabilis* virgin cork had a 48–52 growth ring number, whereas *Q. suber* reproduction cork showed 10 to 11 growth rings. The growth ring width of *Q. variabilis* virgin cork rapidly decreased from the cork back to the 25th growth ring, becoming constant after the 25th growth ring. By contrast, the growth ring width of *Q. suber* reproduction cork gradually decreased from the cork back to the phellogen. Kim [18,23] reported that the average growth ring width in *Q. variabilis* and *Q. suber* reproduction cork was 0.82 mm and 2.06 mm, respectively. The growth ring width of *Q. variabilis* reproduction cork in previous studies was higher than that of virgin cork in the present study.

tion cork in previous studies was higher than that of virgin cork in the present study.

The radial variation in the growth ring width from the cork back to the phellogen is

The radial variation in the growth ring width from the cork back to the phellogen is

shown in Figure 3. *Q. variabilis* virgin cork had a 48–52 growth ring number, whereas *Q. suber* reproduction cork showed 10 to 11 growth rings. The growth ring width of *Q. variabilis* virgin cork rapidly decreased from the cork back to the 25th growth ring, becoming constant after the 25th growth ring. By contrast, the growth ring width of *Q. suber* reproduction cork gradually decreased from the cork back to the phellogen. Kim [18,23] reported that the average growth ring width in *Q. variabilis* and *Q. suber* reproduction cork

shown in Figure 3. *Q. variabilis* virgin cork had a 48–52 growth ring number, whereas *Q. suber* reproduction cork showed 10 to 11 growth rings. The growth ring width of *Q. variabilis* virgin cork rapidly decreased from the cork back to the 25th growth ring, becoming constant after the 25th growth ring. By contrast, the growth ring width of *Q. suber* reproduction cork gradually decreased from the cork back to the phellogen. Kim [18,23] reported that the average growth ring width in *Q. variabilis* and *Q. suber* reproduction cork was 0.82 mm and 2.06 mm, respectively. The growth ring width of *Q. variabilis* reproduction cork in previous studies was higher than that of virgin cork in the present study.

*Forests* **2022**, *13*, x FOR PEER REVIEW 6 of 15

**Figure 3.** Variation in growth ring width from the cork back to the phellogen in *Q. variabilis* virgin cork and *Q. suber* reproduction cork. **Figure 3.** Variation in growth ring width from the cork back to the phellogen in *Q. variabilis* virgin cork and *Q. suber* reproduction cork. and *Q. suber* reproduction cork were 7.41% and 0.97%, respectively. Both species showed an increase in the latecork cell percentage from the near cork back to the near phellogen

The latecork percentage and radial variation in the cork of both species are presented in Table 3 and Figure 4, respectively. The latecork percentages in *Q. variabilis* virgin cork and *Q. suber* reproduction cork were 7.41% and 0.97%, respectively. Both species showed an increase in the latecork cell percentage from the near cork back to the near phellogen stage. Kim [18,23] mentioned that the latecork percentage of *Q. variabilis* was higher than that of *Q. suber,* which was 14.00% in *Q. variabilis* and 10.60% in *Q. suber*. The latecork percentage in the previous study was higher than that in the present study. Pereira et al. The latecork percentage and radial variation in the cork of both species are presented in Table 3 and Figure 4, respectively. The latecork percentages in *Q. variabilis* virgin cork and *Q. suber* reproduction cork were 7.41% and 0.97%, respectively. Both species showed an increase in the latecork cell percentage from the near cork back to the near phellogen stage. Kim [18,23] mentioned that the latecork percentage of *Q. variabilis* was higher than that of *Q. suber,* which was 14.00% in *Q. variabilis* and 10.60% in *Q. suber*. The latecork percentage in the previous study was higher than that in the present study. Pereira et al. [4] also reported that, within a growth ring, *Q. suber* virgin cork showed a higher proportion of latecork cells than *Q. suber* reproduction cork. stage. Kim [18,23] mentioned that the latecork percentage of *Q. variabilis* was higher than that of *Q. suber,* which was 14.00% in *Q. variabilis* and 10.60% in *Q. suber*. The latecork percentage in the previous study was higher than that in the present study. Pereira et al. [**Error! Reference source not found.**] also reported that, within a growth ring, *Q. suber* virgin cork showed a higher proportion of latecork cells than *Q. suber* reproduction cork. **Table 3.** Latecork percentage in *Q. variabilis* virgin cork and *Q. suber* reproduction cork.

[**Error! Reference source not found.**] also reported that, within a growth ring, *Q. suber* **Table 3.** Latecork percentage in *Q. variabilis* virgin cork and *Q. suber* reproduction cork. *Q. variabilis* **(%)** *Q. suber* **(%)** 


*Q. variabilis* **(%)** *Q. suber* **(%)**  Tree 1 Tree 2 Tree 3 Average Plank 1 Plank 2 Average Note: Numbers in parentheses are the SD. The listing of the same superscript lowercase letters beside the mean values in the same row denotes nonsignificant outcomes at the 5% significance level for comparisons between species. side the mean values in the same row denotes nonsignificant outcomes at the 5% significance level for comparisons between species.

**Figure 4.** Variation of latecork percentage from the cork back to the phellogen in *Q. variabilis* virgin cork and *Q. suber* reproduction cork. **Figure 4.** Variation of latecork percentage from the cork back to the phellogen in *Q. variabilis* virgin cork and *Q. suber* reproduction cork.

#### *3.2. Cellular Structure Observations*

cork and *Q. suber* reproduction cork.

*3.2. Cellular Structure Observations* 

**Figure 4.** Variation of latecork percentage from the cork back to the phellogen in *Q. variabilis* virgin *3.2. Cellular Structure Observations*  SEM images in the transverse, radial, and tangential sections of *Q. variabilis* virgin cork and *Q. suber* reproduction cork are displayed in Figure 5. In the transverse and radial sections, the earlycork and latecork cells of both species showed a brick wall-type structure.

and nonagonal.

100 μm.

(0.30)

(0.03)

(0.06)

Sclereid 0.15 a

In the tangential section, the cork cells of both species showed a honeycomb structure with various shapes, such as rectangular, pentagonal, hexagonal, heptagonal, octagonal, and nonagonal. ture. In the tangential section, the cork cells of both species showed a honeycomb structure with various shapes, such as rectangular, pentagonal, hexagonal, heptagonal, octagonal,

cork and *Q. suber* reproduction cork are displayed in Figure 5. In the transverse and radial sections, the earlycork and latecork cells of both species showed a brick wall-type struc-

*Forests* **2022**, *13*, x FOR PEER REVIEW 7 of 15

**Figure 5.** Transverse (**A**,**D**), radial (**B**,**E**), and tangential (**C**,**F**) sections of *Q. variabilis* virgin cork (**A**– **C**) and *Q. suber* reproduction cork (**D**–**F**). Earlycork (Ec) and latecork (Lc). White scale bar represents **Figure 5.** Transverse (**A**,**D**), radial (**B**,**E**), and tangential (**C**,**F**) sections of *Q. variabilis* virgin cork (**A**–**C**) and *Q. suber* reproduction cork (**D**–**F**). Earlycork (Ec) and latecork (Lc). White scale bar represents 100 µm.

The results of previous studies are in line with those reported in the present study. *Q. variabilis* and *Q. suber* corks showed a brick wall-type structure in the transverse and radial sections and a honeycomb structure in the tangential section [4,12,13,16]. The results of previous studies are in line with those reported in the present study. *Q. variabilis* and *Q. suber* corks showed a brick wall-type structure in the transverse and radial sections and a honeycomb structure in the tangential section [4,12,13,16].

The element compositions of *Q. variabilis* virgin cork and *Q. suber* reproduction cork in the three sections are shown in Table 4. *Q. variabilis* virgin cork consisted of cork cells, lenticular channels, dark-brown zones, and sclereids, whereas *Q. suber* reproduction cork consisted of cork cells and lenticular channels. The proportions of cork cells in the transverse, radial, and tangential sections of *Q. variabilis* were 86.85%, 84.06%, and 84.68%, respectively, whereas those of *Q. suber* were 95.45%, 91.43%, and 93.03%, respectively. Both species had a higher proportion of cork cells in the transverse section than in the radial The percentage of cork tissue of *Q. variabilis* virgin cork and *Q. suber* reproduction cork in the three sections are shown in Table 4. *Q. variabilis* virgin cork consisted of cork cells, lenticular channels, dark-brown zones, and sclereids, whereas *Q. suber* reproduction cork consisted of cork cells and lenticular channels. The proportions of cork cells in the transverse, radial, and tangential sections of *Q. variabilis* were 86.85%, 84.06%, and 84.68%, respectively, whereas those of *Q. suber* were 95.45%, 91.43%, and 93.03%, respectively. Both species had a higher proportion of cork cells in the transverse section than in the radial and tangential sections; however, the proportion of cork cells in all the sections of *Q. suber* was significantly higher than that of *Q. variabilis*.


and tangential sections; however, the proportion of cork cells in all the sections of *Q. suber* 

was significantly higher than that of *Q. variabilis*. **Table 4.** Cork tissue percentages in *Q. variabilis* virgin cork and *Q. suber* reproduction cork.

> (0.79) (0.33) - - - 0.17 a 0.41 b Note: Numbers in parentheses are the SD. The listing of the same superscript lowercase letters beside the mean values in the same row denotes nonsignificant outcomes at the 5% significance level for comparisons between sections in both species.

(0.10) - - -

Note: Numbers in parentheses are the SD. The listing of the same superscript lowercase letters beside the mean values in the same row denotes nonsignificant outcomes at the 5% significance level

The lenticular channel proportions in the transverse, radial, and tangential sections of *Q. variabilis* cork were 10.68%, 13.00%, and 12.01%, respectively, whereas those of *Q. suber* were 4.55%, 8.57%, and 6.97%, respectively. The radial section of both species had the highest proportion of lenticular channels among the three sections. Additionally, there was a significant difference in the proportion of lenticular channels in the three sections between the two species.

The proportions of the dark-brown zones in the transverse, radial, and tangential sections were 2.33%, 2.77%, and 2.89%, respectively, whereas those of sclereid were 0.15%, 0.17%, and 0.41%, respectively. The highest proportion of dark-brown zones and sclereids in *Q. variabilis* virgin cork was observed in the tangential section. Additionally, the proportion of dark-brown zones and sclereids was the lowest in the transverse section. There was no significant difference in the proportion of dark-brown zone between sections in *Q. variabilis* cork, whereas the proportion of sclereids in the tangential section showed a significant difference than in the transverse and radial sections.

The results of previous studies are in line with those reported in the present study. Kim [18,23] reported that *Q. variabilis* and *Q. suber* reproduction corks consisted of 87.20% and 93.00% cork cells, 9.00% and 4.60% lenticels, 0.80% and 0.60% sclereids, and 3.00% and 1.80% dark-brown zones. The author also reported a significant difference in the proportion of cork cells and lenticels between the two species. Notwithstanding, no significant difference was observed in terms of the proportion of sclereids and dark-brown zones between the two species. Li et al. [17] reported that the virgin and reproduction corks of *Q. variabilis* showed lenticular channels and sclereids encircled by dark and hard layers. Additionally, lenticular channels and sclereids surrounded by the dark and hard layers were less frequent in reproduction cork compared to virgin cork.

#### *3.3. Cork Cell Dimensions*

#### 3.3.1. Number of Edges in a Cork Cell

The frequency of edge numbers in *Q. variabilis* virgin cork and *Q. suber* reproduction cork are presented in Table 5. In the transverse and radial sections, the cork cells in *Q. variabilis* virgin cork frequently exhibited a hexagonal shape (59.75% and 56.50%, respectively), while pentagonal shape was observed in 19.00% and 19.75% of cells in the transverse and radial sections, respectively. Moreover, 16.75% and 18.50% of cork cells showed a heptagonal shape in the transverse and radial sections, respectively. A small amount of octagonal and rectangular shapes was observed in the transverse section (2.00% and 2.50%, respectively) and radial section (3.75% and 1.50%, respectively). In the tangential section, a hexagonal shape was observed in 50.00% of the cork cells in *Q. variabilis* virgin cork. Pentagonal and heptagonal shapes were also observed in the tangential section (34.00% and 10.75%, respectively). Cork cells also displayed a small percentage of octagonal and rectangular shapes (1.00% and 4.00%, respectively). In addition, the dispersion of the edge number in the tangential section was 67.00%, which was higher than that in the transverse and radial sections (53.75% and 59.25%, respectively).

In *Q. suber*, cork cells frequently showed a hexagonal shape of 57.00% in the transverse section and 55.00% in the radial section. Cork cells in the transverse and radial sections also exhibited pentagonal shape (22.75% and 26.00%, respectively). Moreover, cork cells were heptagonal in shape in the transverse and radial sections (16.25% and 15.75%, respectively). In addition, octagonal and rectangular cork cells were observed in the transverse section (1.75% and 2.25%, respectively) and radial section (1.50% and 1.75%, respectively). In the tangential section, 48.00% of cork cells were hexagonal cell, 26.75% were pentagonal, 19.00% were heptagonal, 4.25% were rectangular, and 1.75% were octagonal. Furthermore, both species showed a small proportion of nonagonal shapes in the tangential section (0.25%). In addition, the dispersion of the edge number in the tangential section was higher than those in the transverse and radial sections (72.00%, 55.00%, and 54.75%, respectively).


**Table 5.** Frequency of the edge number in *Q. variabilis* virgin cork and *Q. suber* reproduction cork.

In the transverse and radial sections, a smaller number of cork cells with a pentagonal shape was observed in *Q. variabilis* virgin cork compared to *Q. suber*. In the tangential section, *Q. variabilis* had a higher proportion of pentagonal cells than *Q. suber*. In addition, *Q. variabilis* cork cells also showed a smaller proportion of heptagonal shapes compared to *Q. suber.* The dispersion of the edge number in the transverse and tangential sections in *Q. variabilis* was smaller than that in *Q. suber*, whereas that in the radial section was higher in *Q. variabilis* than in *Q. suber*. Overall, both cork species showed a comparable distribution of cell shapes in the three sections, in line with the results of previous studies.

Pereira et al. [4] reported that 52.60%, 56.20%, and 47.80% of the cork cells in *Q. suber* virgin and reproduction corks had a hexagonal shape in the transverse, radial, and tangential sections, respectively. These authors also reported that pentagonal and heptagonal cork cells accounted for 22.60% and 17.80% of cork cells in the transverse section, 20.30% and 17.20% in the radial section, and 24.90% and 21.60% in the tangential section, respectively. The dispersion of the number of sides in the tangential section was 71.00%, which was higher compared to those in the transverse and radial sections (70.00%, and 62.00%, respectively). Yafang et al. [16] reported that 53.60%, 52.50%, and 52.30% of virgin cork cells in *Q. variabilis* were hexagonal in shape in the transverse, radial, and tangential sections, respectively. The cork cells in the transverse, radial, and tangential sections also exhibited a pentagonal shape (23.40%, 25.90%, and 22.60%, respectively). The heptagonal shape of the transverse, radial, and tangential sections accounted for 14.90%, 14.30%, and 19.50% of cork cells, respectively. The authors also reported that the dispersion of the number of sides of cork cells in the transverse, radial, and tangential sections were 72.00%, 69.00%, and 73.00%, respectively, which were higher in the tangential section than in the transverse and radial sections. Miranda et al. [12] reported that the number of edges in *Q. variabilis* reproduction cork frequently showed a hexagonal shape in the tangential section, accounting for 60.00% of cork cells. Furthermore, 19.40% and 17.60% of cells exhibited pentagonal and heptagonal shapes in the tangential section, respectively, while 39.50%, 37.10%, 18.50%, and 4.80% exhibited pentagonal, hexagonal, rectangular, and heptagonal shapes in the non-tangential sections, respectively. Additionally, the dispersion of the number of edges in the non-tangential sections was higher than that in the tangential section (74.80% vs. 49.10%, respectively). Ferreira et al. [13] reported that there were 40.50% and 41.20% hexagonal cells, 31.30% and 32.10% pentagonal cells, and 18.20% and 17.70% heptagonal cells in the tangential section of the virgin and reproduction corks of *Q. variabilis*, respectively. The dispersion of the number of cell edges in the tangential section of virgin cork was higher than that of reproduction cork (91.30% vs. 84.60%, respectively). The authors also reported that in the non-tangential sections, virgin cork commonly showed a pentagonal shape (44.40%), whereas reproduction cork generally showed a hexagonal shape (46.90%). The percentages of hexagonal and rectangular shapes were 32.10% and 21.20%, respectively, in virgin cork. In reproduction cork, pentagonal and rectangular shapes were observed in

26.30% and 22.10% of the cork cells, respectively. Virgin cork showed a smaller dispersion of the number of edges of cells in the non-tangential sections than reproduction cork, which was 60.20% and 75.90%, respectively.

3.3.2. Two-Dimensional Characteristics of Cork Cells

The two-dimensional characteristics of the earlycork and latecork cells in *Q. variabilis* virgin cork and *Q. suber* reproduction cork are summarized in Table 6.

**Table 6.** Two-dimensional characteristics of the cork cells in *Q. variabilis* virgin cork and *Q. suber* reproduction cork.


Note: Numbers in parentheses are the SD. The listing of the same superscript lowercase letters beside the mean values in the same row denotes nonsignificant outcomes at the 5% significance level for comparisons between earlycork and latecork in both species.

In *Q. variabilis*, the radial and tangential widths of earlycork cells were higher than those of latecork cells as 15.81 µm and 22.78 µm for earlycork cells and 7.10 µm and 22.62 µm for latecork cells, respectively. There were significant differences in radial width between earlycork and latecork cells, whereas, in tangential width, there was no significant difference between earlycork and latecork cells. For the radial and tangential cell walls thickness: 1.21 µm and 1.14 µm for the earlycork cells and 1.45 µm and 1.53 µm for the latecork cells. The radial and tangential cell walls were significantly thicker in the latter than in the earlycork cells. Earlycork and latecork cells showed tangential lumen diameter of 21.84 µm and 21.10 µm, respectively. The radial lumen diameter was greatly higher in the earlycork cells than in the latecork cells showing 14.59 µm and 5.66 µm, respectively. The difference between earlycork and latecork cells in radial and tangential lumen diameters was statistically confirmed.

In *Q. suber*, the radial width of earlycork and latecork cells was 37.61 µm and 14.33 µm, respectively, while the tangential width of earlycork and latecork cells was 27.16 µm and 26.16 µm, respectively. The radial width of the earlycork cells was greater than that of the latecork cells, while the tangential width of earlycork cells was slightly larger than that of latecork cells. The difference between earlycork and latecork cells in radial and tangential widths was statistically significant. The thickness of radial and tangential walls was 1.50 µm and 1.45 µm for earlycork cells and 2.00 µm and 2.31 µm for latecork cells, respectively. Both cell walls were significantly thicker in the latecork cells than in the earlycork cells. The tangential lumen diameter of earlycork cells was slightly larger than that of latecork cells (25.36 µm and 23.81 µm, respectively), whereas the radial lumen diameter of earlycork cells was larger than that of latecork cells (35.90 µm and 12.17 µm, respectively). The difference between earlycork and latecork cells in radial and tangential lumen diameters was statistically confirmed. Additionally, the earlycork and latecork cells in *Q. suber* reproduction cork were significantly larger in cell width and lumen diameter and thicker in cell wall thickness than those in *Q. variabilis* virgin cork.

In this study, differences were observed in the two-dimensional characteristics between species and between the earlycork and latecork cells in each species, in line with previous studies. Pereira et al. [4] reported that the radial width of the cork cells of *Q. suber* was 30.00–40.00 µm in the earlycork cells and 10.00–15.00 µm in the latter. The authors also mentioned that the radial cell wall thickness in the earlycork cells was 1.00–1.50 µm and approximately twice as large in the latecork cells (2.00–3.00 µm). Kim [18] found that the radial width in the reproduction cork cells of *Q. variabilis* from Korea and *Q. suber* from Portugal were 26.30 µm and 44.20 µm in the earlycork cells and 6.90 µm and 10.60 µm in the latecork cells, accordingly. The author also found that the cork cell walls in *Q. variabilis* and *Q. suber* were 0.60 µm and 1.00 µm in the earlycork cells and 1.00 µm and 1.80 µm in the latecork cells. Yafang et al. [16] reported that the radial width of *Q. variabilis* virgin cork was 15.00–35.00 µm in the earlycork cells and 10.00 µm in the latter. These authors also mentioned that the earlycork cells showed smaller radial cell walls thickness than the latecork cells (1.00–1.50 µm vs. 3.00 µm). Miranda et al. [12] reported that earlycork cells in *Q. variabilis* reproduction cork from China showed a larger radial width than latecork cells (21.40 µm vs. 10.40 µm). The authors also reported that the earlycork cells showed smaller radial cell walls thickness than the latecork cells (1.20 µm vs. 2.80 µm). Ferreira et al. [13] also reported that the radial width of earlycork cells in *Q. variabilis* virgin cork was smaller than that in reproduction cork (19.20 µm vs. 22.70 µm, respectively).

3.3.3. Three-Dimensional Characteristics of Cork Cells

The three-dimensional characteristics of the cork cells in *Q. variabilis* virgin cork and *Q. suber* reproduction cork are presented in Table 7. The prism base edge of the cork cells in *Q. variabilis* virgin cork was smaller than that in *Q. suber* reproduction cork (14.81 µm vs. 15.88 µm). The aspect ratio of prism height to prism base edge in *Q. variabilis* virgin cork was 1.07 for earlycork cells and 0.48 for latecork cells, while that in *Q. suber* reproduction cork was about 2.37 for earlycork cells and 0.90 for latecork cells. The prism base area in the earlycork cells of *Q. variabilis* was smaller than that of *Q. suber* (623 µm<sup>2</sup> vs. 830 µm<sup>2</sup> ). Significant differences were observed in the prism base edge and area of earlycork cells between the two species.


**Table 7.** Three-dimensional characteristics of the cork cells in *Q. variabilis* virgin cork and *Q. suber* reproduction cork.

Note: Numbers in parentheses are the SD. The listing of the same superscript lowercase letters beside the mean values in the same row denotes nonsignificant outcomes at the 5% significance level for comparisons between earlycork and latecork in both species.

The cork cell volume in *Q. variabilis* was 1.27 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for earlycork cells and 0.57 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for latecork cells, while that in *Q. suber* was 3.47 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for earlycork cells and 1.32 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for latecork cells. In both species, the cork cell volume of earlycork cells was larger than that of latecork cells. Additionally, significant differences were observed in the volume of earlycork and latecork cells between species.

The solid volume in *Q. variabilis* was 0.21 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for earlycork cells and 0.17 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for latecork cells, whereas that in *Q. suber* was 0.49 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for earlycork cells and 0.34 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> for latecork cells, which was significantly bigger than those in *Q. variabilis*. The solid volume of latecork cells in both species was smaller than that of earlycork cells, which was statistically confirmed.

The fractional solid volume of the cork cell in *Q. variabilis* was 30.75% for latecork cells and 16.40% for earlycork cells, while that in *Q. suber* was 26.39% for latecork cells and 14.29% for earlycork cells. The fractional solid volume in *Q. suber* was significantly smaller than that in *Q. variabilis*. The volume of latecork cells in both species was nearly double that of earlycork cells, showing significant difference between species.

*Q. variabilis* virgin cork had 7.86 <sup>×</sup> <sup>10</sup><sup>7</sup> earlycork cells per cm<sup>3</sup> and 17.50 <sup>×</sup> <sup>10</sup><sup>7</sup> latecork cells per cm<sup>3</sup> , whereas *Q. suber* reproduction cork *had* 2.88 <sup>×</sup> <sup>10</sup><sup>7</sup> earlycork cells per cm<sup>3</sup> and 7.60 <sup>×</sup> <sup>10</sup><sup>7</sup> latecork cells per cm<sup>3</sup> , respectively. *Q. variabilis* virgin cork had more cork cells per cm<sup>3</sup> than *Q. suber* reproduction cork.

Kim [18] reported that *Q. variabilis* and *Q. suber* reproduction corks showed no difference in terms of the prism edge length between earlycork and latecork cells. The prism edge length of *Q. variabilis* was smaller than that of *Q. suber*. These results are in line with those reported in the present study. In a previous study, Yafang et al. [16] reported that the prism base edge and the average base area of *Q. variabilis* virgin cork were 8.00–14.00 µm and 200–500 µm<sup>2</sup> in the earlycork cells. Miranda et al. [12] reported that the prism base edge and the average base area of *Q. variabilis* reproduction cork were 17.20 µm and 764 µm<sup>2</sup> , respectively, while the aspect ratio between prism height and prism base edge in earlycork cells was approximately 1:10. The authors also reported that the total cell volume of the earlycork cells was 1.60 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> , which was higher than that of the latecork cells (0.80 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> ). Earlycork cells showed a much smaller solid volume fraction than latecork cells (13.10% vs. 40.00%). Ferreira et al. [13] examined earlycork cells in the virgin and reproduction corks of *Q. variabilis* and found that the prism base edge in virgin cork was higher than that in reproduction cork (14.30 µm vs. 13.60 µm). Virgin cork also had a higher average base area than reproduction cork (532 µm<sup>2</sup> vs. 477 µm<sup>2</sup> ). In contrast, reproduction cork had a higher total cell volume than virgin cork (1.08 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> vs. 1.02 <sup>×</sup> <sup>10</sup>−<sup>8</sup> cm<sup>3</sup> ). The solid volume fraction of reproduction cork was 14.70%, while that of virgin cork was 11.50%. The three-dimensional characteristics of *Q. variabilis* virgin cork reported in this study were comparable to those of *Q. variabilis* virgin cork reported by Yafang et al. [16] and Ferreira et al. [13]. In addition, the three-dimensional characteristics of reproduction cork reported by Miranda et al. [12] were higher than those of virgin cork in the present study.

#### **4. Conclusions**

*Q. variabilis* virgin cork exhibited a narrower growth ring than *Q. suber* reproduction cork. The latecork percentage of *Q. variabilis* was significantly higher than that of *Q. suber*. *Q. variabilis* cork had a smaller proportion of cork cells and a higher proportion of lenticular channels in the three sections than *Q. suber* cork. Sclereid and dark-brown zones were only observed in *Q. variabilis* cork.

In both species, hexagonal cell shape was commonly observed in all three sections (50–60%). The frequency of pentagonal and heptagonal cell shapes was also higher than that of rectangular, octagonal, and nonagonal shapes in both species.

The cork cells of *Q. variabilis* had smaller cell width, lumen diameter, and cell wall thicknesses than those of *Q. suber*. The earlycork cells of both species had larger radial cells widths and lumen diameters than the latter, while, in both species, the tangential cells width and lumen diameter of earlycork cells were slightly higher than those of the latecork cells.

The prism base edge and area, cork cell volume, and solid volume of the cell wall in *Q. suber* cork were significantly greater than those of *Q. variabilis* cork, whereas the fractional solid volume and number of cells per cm<sup>3</sup> in *Q. variabilis* cork were significantly greater than those of *Q. suber* cork. In both species, earlycork cells showed higher solid and total cell volumes than latecork cells; nevertheless, the fractional solid volume and number of cells per cm<sup>3</sup> in the latecork cells were greater than those in the earlycork cells.

In conclusion, *Q. variabilis* virgin cork grown in Korea shows distinctive quantitative anatomical characteristics compared to *Q. suber* reproduction cork grown in Portugal. Due to the structural characteristics of *Q. variabilis* virgin cork, applications require trituration to cork granules and agglomeration to produce cork composite products, while its cellular features allow considering it for insulation, surfacing, and sealant products. The results of this study may be used to evaluate the quality and identify *Q. variabilis* virgin cork grown in Korea for further utilization.

**Author Contributions:** Conceptualization, D.P. and N.-H.K.; methodology, D.P., B.D.P., J.-H.K., J.- H.J., G.-U.Y., S.-Y.P., S.-H.L. and N.-H.K.; software, D.P.; validation, N.-H.K.; formal analysis, D.P.; investigation, D.P.; resources, N.-H.K.; data curation, D.P.; writing—original draft preparation, D.P.; writing—review and editing, D.P., B.D.P., S.-H.L. and N.-H.K.; visualization, D.P.; supervision, N.- H.K.; project administration, J.-H.K.; and funding acquisition, N.-H.K., S.-H.L. and J.-H.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Science and Technology Support Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) (NRF-2019K1A3A9A01000018 and No. 2022R1A2C1006470), Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2016R1D1A1B01008339 and No. 2018R1A6A1A03025582), and R&D Program for Forest Science Technology (Project No. 2021350C10- 2223-AC03) provided by the Korea Forest Service (Korea Forestry Promotion Institute).

**Data Availability Statement:** The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

