Comparative morphology and anatomy of Korean Boehmeria achenes and their taxonomic significance
Article information
Abstract
This study confirmed the taxonomic significance of achenes by observing and comparing the morphology and anatomical characteristics of 13 taxa of the genus Boehmeria in Korea. The results confirmed that the achenes studied here are the drupe-like fruit type with mucilage in the exocarp, and during the fruiting season, the perianth persists on the pericarp. The morphological characteristics of the achenes, specifically the number per glomerule as well as the size, shape, color, and trichome were taxonomically significant. The anatomical characteristics of the transverse sectional shape and wings, the thickness of each pericarp layer, the shape of the crystalliferous cells in the mesocarp, and the number of sclereid cell layers in the endocarp were also found to be important. Particularly, the size and wings of the achenes were informative for distinguishing between Sect. Tilocnide and Sect. Duretia, while other characteristics were differentiated among series or certain taxa within the series. The taxonomic significance of the achenes is discussed through comparisons with previously studied taxa and their characteristics. Accordingly, the results of this study can provide valuable insight for those seeking to understand the evolutionary trends of achenes in Boehmeria in the future.
INTRODUCTION
The genus Boehmeria Jacq. (Urticaceae Juss.) is one of the largest genera within the family (Gaudichaud, 1830; Weddell, 1856, 1869; Yahara, 1990; Wilmot-Dear and Friis, 2013). This genus is widely distributed in tropical and subtropical regions and rarely in temperate regions (Chen et al., 2003). It comprises approximately 47–120 species, although the number of recognized species varies among authors (Chen et al., 2003; Zhao et al., 2003; Wilmot-Dear and Friis, 2013; Liang et al. 2020). In particular, Northeast and Southeast Asia are known as the centers of species diversity, with approximately 44–75 species distributed in these regions (Satake, 1936; Hutchinson, 1967; Wang, 1981a, 1981b; Yahara, 1981, 1990; Zhao et al., 2003; Pierozzi et al., 2008). The genus Boehmeria was first named by Jacquin (1760) based on the type species B. ramiflora Jacq. from Central America. The generic name is derived from the name of the German botanist Georg Rudolph Böhmer (1723–1803) (Miller, 1971; Wilmot-Dear and Friis, 1996). This genus is characterized by a lack of stinging trichomes, filiform stigmas, persistent stigmas during the fruiting season, perianth remaining on the achenes, scarious achenes, and the lack fleshy receptacles (Chen et al., 2003; Tateishi, 2006; Kim, 2018).
The classification system of this genus was first established by Gaudichaud (1830), who classified the genus Boehmeria within the family Urticaceae, tribe Boehmerieae, based on morphological characteristics such as phyllotaxis, flower shape, and achene shape. Weddell (1854) classified the Urticaceae into three tribes: Boehmerieae, Lecantheae, and Urereae. The tribe Boehmerieae was further divided into five subtribes, and Gaudichaud’s (1830) classification system was revised by subdividing the subtribe Euboehmerieae into six genera: Boehmeria, Chamabainia, Cypholophus, Didymogyne, Margarocarpus, and Pouzolzia. Blume (1857) classified 74 species of Boehmeria based on key characteristics such as the phyllotaxis, leaf margin shape, leaf texture, inflorescence shape, presence of presence of a stipitate ovary, style shape, and achene shape, into six sections: Chamabainia, Duretia, Genuinae, Margarocapus, Tilocnide, and Urocnide. Weddell (1869) classified the subtribe Euboehmerieae into four genera: Boehmeria, Chamabainia, Memorialis, and Pouzolzia. Additionally, 47 species and 37 varieties of Boehmeria were categorized into four groups based on phyllotaxis and inflorescence shape. Satake (1936) classified 40 species, 5 varieties, and 4 forms of Japanese Boehmeria based on phyllotaxis into the subgenera Duretia and Tilocnide. The subgenus Duretia was further subdivided into three groups based on shape and trichome of achenes: group 1 (sect. Densiflorae, Spicatae, and Zollingerianae), group 2 (sect Sieboldianae), and group 3 (sect. Longispicae, Pannosae, and Splitgerbera). Wang (1981a, 1981b) examined evolutionary trends in morphological characteristics of 55 Chinese Boehmeria taxa, including habitat, phyllotaxis, lobed shape of the leaf apex, inflorescence shape, stamen number in male flowers, presence of pedicels in male flowers, presence of stipes on fruits, and presence of wings on achenes. Based on these results, the taxa were classified into five sections: Boehmeria, Duretia, Phyllostachys, Tilocnide, and Zollingerianae. The section Duretia was further divided into four series—Densiflorae, Ingjiangenses, Longispicae, and Siamenses—based on characteristics such as leaf shape, inflorescence shape, presence of pedicels, achene shape, and presence of wings on achenes. Relationships among the sections and series were also presented. Afterward, Wang (2016) established a new series, Spicatae, within the section Duretia. This series exhibited distinct differences from the taxa of the series Longispicae within the same section in morphological characteristics such as plant trichomes, stem color, stem diameter, leaf size, leaf texture, leaf trichomes, inflorescence shape, and trichomes on female flowers. Tateishi (2006) supplemented Satake’s (1936) classification system and classified 21 taxa of Japanese Boehmeria into 2 subgenera (Boehmeria and Tilocnide) and 5 sections (Densiflorae, Longispicae, Sieboldianae, Spicatae, and Splitgerbera) based on characteristics such as habitat, stem color, phyllotaxis, leaf margin shape, presence of white trichomes on the abaxial surfaces of leaves, leaf texture, achene shape, and achene trichomes. Since the classification system for this genus was established by Gaudichaud (1830), it has been primarily refined based on taxa from East and Northeast Asia, including those from China and Japan. To this day, many authors continue to adhere to the traditional classification system based on morphological studies. Additionally, in several phylogenetic studies, the infrageneric classification of Boehmeria is treated at the section level rather than the subgenus level (Guo, 1999; Kang et al., 2008; Liang, 2009; Yu et al., 2015; Liang et al., 2020). Furthermore, the genus Boehmeria is known to be polyphyletic within the family Urticaceae (Wu et al., 2013).
Satake (1936) conducted a comparative study of petiole anatomical characteristics in Japanese Boehmeria. Wilmot-Dear and Friis (1996) and Wilmot-Dear et al. (2009) clearly distinguished between the genera Pouzolzia and Boehmeria through morphological and anatomical comparisons of their achenes. Kravtsova et al. (2000) conducted morphological and anatomical studies on the achenes of nine endemic species in the New World. They classified the achenes into two types: an achene-like fruit with a thin and dry exocarp and a drupe-like fruit with a fleshy and mucilaginous exocarp. Additionally, based on anatomical characteristics, such as mucilage and tannins in the exocarp, crystalliferous cells in the mesocarp, and pericarp ribs, they categorized into four groups, and evolutionary trends were inferred accordingly. They also proposed the necessity of morphological and anatomical studies of achenes for the Old World taxa. To date, no studies have been conducted on the genus Boehmeria in the Old World, including Korea. Various studies have idenditified the achene in Urticaceae as a taxonomically significant characteristic (Kravtsova et al., 2020).
Therefore, this study aimed to compare the morphological and anatomical characteristics of Korean Boehmeria achenes to confirm their taxonomic significance. This study focuses on 13 taxa of Korean Boehmeria, which have not been previously studied (Jo, 2023, Jo et al., 2021, 2023; Korea National Arboretum, 2023), and the classification system follows that of Wang (1981a, 1981b, 2016).
MATERIALS AND METHODS
Materials
This study investigated 13 taxa of Boehmeria distributed in Korea (Table 1). The achenes were collected during the fruiting seasons between 2018 and 2022, and both specimens preserved in 70% ethanol and dry specimens were used. The voucher specimens used in the study were deposited at the Herbarium of Andong National University (ANH). In Boehmeria, achene trichomes have not been clearly defined, and authors describe them differently (Lee, 1996; Lee, 2003; Chen et al., 2003; Lee, 2006; Tateishi, 2006; Kim, 2018). Therefore, the terminology for trichomes was referenced from specialized publications, such as Harris and Harris (2001), Simpson (2019), and Beentje (2020) (Fig. 1).
Micromorphological structure
Achenes fixed in 70% ethanol were dehydrated stepwise in 80–100% ethanol for 30 min each to observe and photograph the micromorphological structures. Impurities were removed using an ultrasonic cleaning bath (2510 EDTH, Branson, Danbury, CT, USA) after each solution substitution. Afterward, the achenes were dehydrated by substituting liquid carbon dioxide 18 times using a critical point dryer (Leica EM CPD 300, Leica Microsystems, Wetzlar, Germany). Subsequently, they were coated with gold (Au) for 65 seconds using a sputter coater (108 auto, Cressington Scientific Instruments Ltd., Watford, UK). The coated achenes were observed and photographed using a field emission scanning electron microscope (FE-SEM; MIRA 3 XMH, Tescan, Brno, Czech Republic). SEM images of achenes were acquired using 15 keV, an SE detector, and a working distance of 30 to 70 mm (Fig. 2).
Transverse section structure
Transverse sections of the achenes were observed in mature individuals selected from the same population size. The midpoint of the achenes, preserved in 70% ethanol, was excised and washed twice for 1 h each in 50% ethanol. Subsequently, they were progressively dehydrated in 10–100% tert-butyl alcohol and embedded twice for 12 h each in paraffin (Leica TP1020, Leica Biosystems, Wetzlar, Germany). The samples were then made into paraffin blocks (Leica EG1160, Leica Biosystems, Wetzlar, Germany) and sectioned at a thickness of 12 μm using a microtome (Leica RM 2155, Leica Biosystems, Wetzlar, Germany). These sectioned samples were affixed to microscope slides using a solution of albumin and distilled water and dried for over 4 h on a hot plate (HP630D, MTOPS, Seoul, Korea). The slides were double-stained with safranin and fast green. Finally, the samples were mounted in toluene to prepare permanent microscope slides. They were observed and photographed using an optical microscope (light microscope [LM]; AX-70, Olympus, Tokyo, Japan) with a microscope digital camera (DP2-BSW, Olympus, Hamburg, Germany). The captured images were then measured and analyzed using i-solution lite software (IMT i-Solution Inc., Daejeon, Korea) (Fig. 3).
RESULTS
Morphological and micromorphological characteristics of achenes
The infructescences of Korean Boehmeria are attached to the upper and middle parts of the stems. Basic infructescences consist of glomerules composed of clusters of multiple sessile or subsessile achenes, and these glomerules are arranged in a spike (Fig. 4).
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Number of achenes per glomerule
The number of achenes per glomerule in B. holosericea can reach up to 396, whereas B. sieboldiana has the fewest among related taxa, with a minimum of 13 (Fig. 5, Table 2). The wider the width of the glomerule, the more achenes are clustered together. In particular, B. holosericea, B. japonica, B. platanifolia, and B. quelpaertensis tend to have more clustered achenes compared to related taxa.
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Size and shape
The average size of the achenes in B. holosericea and B. quelpaertensis is observed to be the largest, while they are the smallest in B. nivea and B. nivea var. nipononivea (Table 2). Achenes exhibit various shapes due to the characteristics of their glomerules. However, based on the shapes most commonly observed, they are classified into four types. The obovoid type is observed in B. nivea and B. nivea var. nipononivea; the narrowly obovoid type is observed in B. holosericea and B. quelpaertensis; the sub-orbicular type is observed in B. hirtella and B. nakashimae, and the remaining taxa have rhomboid to obovoid types (Fig. 2, Table 2). The base shape of the achenes typically appears cuneate in most taxa. However, in B. holosericea and B. quelpaertensis, it is observed as narrow cuneate or attenuate, distinctly distinguishing them from other taxa (Fig. 2C, F, Table 2).
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Color
The color of the achenes ripens from yellow-green to brown in all taxa. However, the achenes of B. sieboldiana, B. gracilis, B. paraspicata, and B. silvestrii are generally red. Nevertheless, like other taxa, they turn brown when fully mature (Table 2).
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Trichomes
The trichomes of achenes are dense in most taxa. However, B. nivea var. nipononivea is relatively sparse compared to its closely related taxa. This characteristic is very important for distinguishing most taxa within the genus. The hirsute type is observed in B. nivea and B. platanifolia, the hirsutulous type in B. japonica and B. quelpaertensis, the strigillose type in B. nipononivea, B. hirtella, B. nakashimae, and B. sieboldiana, the sericeous type in B. holosericea, and the scabrous type in B. gracilis, B. paraspicata, B. silvestrii, and B. spicata (Figs. 1, 2, Table 2). Additionally, each type of trichome is composed of both sparsely distributed uncinate bristles and glandular trichomes, with numerous protuberances covering the surfaces of the trichomes.
Anatomical characteristics of achenes
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Shape of the transverse section
The transverse section is observed in three shapes: narrowly elliptic, elliptic, and broadly elliptic. Specifically, B. holosericea exhibits a narrowly elliptic shape, B. nivea and B. nivea var. nipononivea exhibit a broadly elliptic shape, and the remaining taxa exhibit an elliptic shape (Fig. 3, Table 3). Wings on the achenes are observed in all taxa except B. nivea and B. nivea var. nipononivea, with both taxa having wings that are nearly absent on both sides of the achenes (Fig. 3A, B, Table 3). In contrast, distinct wings develop in the remaining taxa. A developed vascular bundle appears on each side of the wing.
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Fruiting perianth
In all taxa, the perianths developed from the flowering season persist to the fruiting stage. These perianths are thin, membranous, and completely surround the pericarp, to which they are closely attached (Fig. 3). The perianth is observed as orbicular, rectangular, or polygonal during early fruiting stages. However, when fully mature, the two perianth layers form flattened cells due to contraction (Fig. 3, Table 2). The outer surface is covered with unicellular trichomes with one to three layers of subsidiary cells at the base of the trichomes, which are sparse or dense depending on the taxon. Within the perianth layers, several calcium oxalate crystals of the druse type are sparsely distributed.
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Exocarp
The exocarp appears as a single layer in all taxa, surrounding the endocarp (Fig. 3). The average thickness of B. quelpaertensis is observed to be the thickest at 81.84 μm, while B. holosericea is confirmed to be the thinnest at an average of 36.29 μm (Fig. 3C, F, Table 3). The exocarp contains mucilage cells that secrete viscous substances, and the cells, excluding the wing portion, are observed in a rounded rectangular to rounded square shape or transversely rounded rectangular to rounded square shape (Fig. 3). The mucilage within these cells appears stained red or purple by safranin-fast green, and fully dried seeds do not exhibit mucilage. Depending on the taxa, tannin cells containing pigments develop within the mucilage cells. In this study, tannin cells are only observed in B. quelpaertensis, B. japonica, and B. spicata, but in some cases, these taxa do not have tannin cells (Fig. 3).
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Mesocarp
The mesocarp appears as a single layer in all taxa, with the thickest observed in B. sieboldiana at 30.79 μm and the thinnest in B. silvestrii at 10.11 μm (Table 3). Within the mesocarp, crystalliferous cells form a single layer surrounding the endocarp. These crystalliferous cells are observed as wedge-shaped only in B. hirtella, B. nakashimae, and B. sieboldiana (Fig. 3G–I), and are not observed as wedgeshaped in the remaining taxa. The crystalliferous cells contain irregularly prismatic crystals.
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Endocarp
The endocarp appears as a single layer in most taxa but is observed as 1 to 2 layers in B. hirtella and B. sieboldiana (Fig. 3G, I, Table 3). The average thickness of these two taxa is approximately twice that of their closely related taxa, with averages of 27.93 μm and 35.29 μm, respectively. In contrast, B. spicata is the thinnest at 12.39 μm (Table 3). The endocarp, unlike other pericarp layers, is composed of sclereids with thick lignified secondary walls. These sclereids are generally flattened, rounded, and rectangular to circular, with a rounded or elongated cavity in the center.
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Seed coat and embryo
The seed coat, which surrounds the endosperm, embryo, and cotyledon, is located inside the endocarp, and its thickness is observed to be the greatest in B. quelpaertensis at 14.52 μm and the thinnest in B. nivea var. nipononivea at 8.99 μm (Fig. 3B, F, Table 3). In the achenes within glomerule, the orientation of the cotyledons relative to the carpel varies and includes vertical, diagonal, and horizontal directions (Fig. 3).
DISCUSSION
Based on the results of morphological and micromorphological studies, the number of achenes per glomerule, size, shape, color, and trichome of the achenes are identified as important characteristics in Korean Boehmeria. The present anatomical study reveals that the shape of the transverse section, the presence of wings, the thickness of each pericarp layer, the shape of crystalliferous cells, and the number of endocarp layers are taxonomically useful characteristics. According to the views of Kravtsova et al. (2000) and Kravtsova (2015), the achenes of Korean Boehmeria belong to drupe-like fruit. Additionally, the perianths from the flowering season are confirmed to persist to the fruiting stage and remain attached to the pericarp.
Morphological and micromorphological characteristics of achenes
The number of achenes per glomerule is highest in B. holosericea and lowest in B. sieboldiana (Fig. 5, Table 2). B. holosericea has wide glomerules with narrow spacing between them, whereas B. sieboldiana has narrow glomerules with wide spacing between them. Therefore, the number of achenes is determined by the density and width of the glomerules. Taxa within the Ser. Longispicae tend to have more achenes compared to other taxa. This characteristic is confirmed to be important for distinguishing some taxa.
The shape of the achenes is considered one of the characteristics for dividing series within Sect. Duretia in Wang’s classification system (1981a, 1981b). However, it is confirmed to be useful for initially distinguishing some taxa within the genus. The average size of Sect. Tilocnide is significantly smaller than that of Sect. Duretia, making it an important characteristic for distinguishing between these sections (Fig. 2, Tables 1, 2). While achene size has not been mentioned in any classification system, it is evident that it serves as a distinguishing characteristic within the two sections distributed in Korea. Additionally, in Ser. Longispicae, B. holosericea, and B. quelpaertensis have noticeably longer lengths, and their base shapes are narrowly cuneate or attenuated compared to their closely related taxa (Fig. 2C, F, Tables 1, 2). These two taxa have numerous achenes densely arranged within a single glomerule, which likely influences the length and base shape of the achenes. Therefore, the size and base shape of the achenes are valuable characteristics for classifying certain taxa within a section or series.
The color of the achenes in all taxa changes from yellow-green to brown when fully mature; however, in B. sieboldiana of Ser. Sieboldianae and the taxa of Ser. Spicatae, the achenes commonly change from red to brown (Tables 1, 2). This color difference is attributed to the production of anthocyanin, which significantly increases when exposed to strong sunlight (Gould, 2004; Gould et al., 2010). Anthocyanin production is known to occur in various plant parts, such as stems and leaves (Naing and Kim, 2018), serving as a protective mechanism against light or stress (Gould, 2004; Gould et al., 2010; Naing and Kim, 2021). The taxa mentioned in these two series are believed to produce anthocyanin mainly because they grow in areas with strong sunlight. However, despite growing in environments without shade, no red achenes are observed in B. nivea, B. nivea var. nipononivea, and B. holosericea, suggesting that other factors may be involved. In conclusion, the color of the achenes is considered to be a somewhat important characteristic for distinguishing some taxa.
The morphology of the achenes is confirmed to be a highly important characteristic for distinguishing between taxa (Fig. 2, Table 2). Particularly, Ser. Sieboldianae and Ser. Spicatae are distinguished from closely related series by the strigillose and scabrous trichomes, respectively. However, various types of trichomes are observed among taxa within other sections or series, suggesting rapid divergent evolution processes. The density of trichomes is not confirmed as a distinct characteristic for identification, but it is considered somewhat useful for certain taxa.
Anatomical characteristics of achenes
The shape of the transverse section is useful for distinguishing some taxa. Particularly, within Sect. Duretia, B. holosericea is distinguished from related taxa by its narrowly elliptic shape (Fig. 3C, Tables 1, 3).
The wings of the achenes are presumed to develop by elongation of the exocarp toward the wing. Sect. Duretia shows distinct elongation, while Sect. Tilocnide appears not to be elongated, which is an important characteristic for distinguishing between the sections (Fig. 3A, B, Tables 1, 3). The vascular bundle on each side of the wings of the achenes suggests that it may be involved in wing formation. Therefore, the wings of the achenes are identified as an important characteristic for distinguishing sections, consistent with the classification systems of Wang (1981a, 1981b).
In the family Urticaceae, the fruiting perianth is an important characteristic for distinguishing between genera (Kravtsova et al., 2020). Fruiting perianth adhering to the pericarp is a characteristic observed in some taxa within the genus Boehmeria, and it is known to be useful for distinguishing between Pouzolzia and Pipturus genera (Kravtsova et al., 2000, 2003; Wilmot-Dear et al., 2009). However, it is not considered a key characteristic in the Korean Boehmeria because the perianth is closely attached to the pericarp (Fig. 3). Thus, further research is needed for more taxa, including neighboring countries.
The pericarp structure is regarded as the most important characteristic in systematic studies of taxa within Urticaceae (Kravtsova et al., 2020). Based on this study, the cell layers of the exocarp, mesocarp, and endocarp are each observed as a single layer in all taxa. Therefore, they cannot be applied as taxonomic characteristics (Fig. 3, Table 3). The thickness of the exocarp varies noticeably among some taxa, and this thickness is unrelated to the presence of mucilage cells or tannin cells. Mucilage cells in the exocarp are a characteristic observed only in certain species within some genera of the Urticaceae (Ramstad, 1954; Ryding, 1992; Friis, 1993; Kravtsova et al., 2000, 2020). However, in this study, mucilage cells are observed in all taxa. These mucilage cells are known to develop for seed dispersal or protection (Lobova et al., 2003; Tsai et al., 2021). According to the hypothesis proposed by Lobova et al. (2003), mucilage cells are considered to engage in a biotic interaction that inhibits germination by hindering oxygen absorption when the seeds are submerged in water. The taxa of the Korean Boehmeria mainly grow in moist areas near the water’s edge, supporting the idea that their achenes are dispersed by water. Tannin cells play an important role in protecting plants from animals (McNair, 1930; Evert, 2006). The presence of tannin cells varies among taxa in the New World (Kravtsova et al., 2000). In this study, tannin cells were observed in all taxa. However, even within the same taxa, tannin cells may not be observed depending on the maturity stage of the individual and the achene.
The mesocarp’s thickness also shows noticeable differences in some taxa, similar to the exocarp. The number of crystalliferous layers in the mesocarp is recognized as a significant characteristic. Most taxa from the New World exhibit multiple layers, while some taxa have single-layered structures (Kravtsova et al., 2000). The taxa of Korean Boehmeria from the Old World consistently appear as single-layered structures and are not regarded as a major characteristic (Fig. 3, Table 3); however, the crystalliferous cells of the wedge shape are only observed in Ser. Sieboldianae and are considered useful for distinguishing between series (Fig. 3G–I, Tables 1, 3).
Only B. hirtella and B. sieboldiana of Ser. Sieboldianae exhibit up to two layers of sclereids in the endocarp, making them the thickest among all taxa (Fig. 3G, I, Tables 1, 3). These cells typically provide structural support similar to fibers, but their exact role in certain tissues remains unclear (Evert, 2006; Simpson, 2019). However, sclereids are speculated to aid in deterring herbivory or assist in moisture retention in halophytes, suggesting that these taxa may also serve similar functions (Grigore et al., 2014; Grigore and Toma, 2017; Simpson, 2019).
The thickness of the seed coat varies among some taxa, but most taxa exhibit nearly similar thicknesses and are not recognized as useful characteristics (Fig. 3, Table 3). According to Kravtsova et al. (2000), in most species from the New World, the cotyledons of the embryo were observed to be parallel to the carpel, although, in some species, they were exhibited various orientations. In this study, the cotyledons of each taxon also are exhibited various orientations, such as vertical, diagonal, and horizontal (Fig. 3). Therefore, they are confirmed to have no significance as a taxonomic characteristic.
In conclusion, the achene has been considered an important characteristic for classifying sections (Blume, 1857; Satake, 1936; Wang, 1981a, 1981b; Tateishi, 2006). This study also indicates that some morphological and anatomical characteristics are useful for distinguishing between sections and series. Among the morphological and anatomical characteristics of the achenes, the size and wings of the achenes are identified as characteristics for distinguishing sections. Characteristics such as achene number, shape, color, trichomes, transverse section shape, pericarp thickness, endocarp layer, and seed coat thickness are valuable for distinguishing sections or certain taxa within those sections. Although it is difficult to understand the evolutionary trends of achenes in Korean Boehmeria, this data could provide a valuable foundation for comprehending the evolutionary trends or taxonomic groups of achenes within Urticaceae. Furthermore, it is expected that clear systematics or phylogeny can be inferred when all taxa within the genus Boehmeria (Urticaceae) are examined in future studies.
Acknowledgements
This study was carried out with the support of ‘R&D Program for Forest Science Technology (2021400D10-2225DCA02)’ provided by Korea Forest Service (Korea Forestry Promotion Institute).
Notes
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.