﻿Leaf epidermal micromorphology of Zingiber (Zingiberaceae) from China and its systematic significance

﻿Abstract Leaf epidermal characteristics are important for phylogenetic and taxonomic studies of many plants, but there is currently insufficient such data for this application in Zingiber species. Therefore, the leaf epidermal micromorphology of 22 species in three sections of Zingiber was investigated by light microscopy and scanning electron microscopy. Differences between various taxonomic groups of Zingiberaceae were also compared to assess their phylogenetic and taxonomic significance. As in other genera of Zingiberaceae, the epidermal cells in both the adaxial and abaxial epidermis of Zingiber species were found to be hexagonal or polygonal, with non-sinuous anticlinal walls that are arranged parallel to leaf veins. Tetracytic stomata are mostly randomly distributed in the intercostal regions of both surfaces and are more common on the abaxial surface. The stomatal density of the species in sect. Pleuranthesis is significantly lower than that in sects. Zingiber and Cryptanthium. There are two types of trichome in Zingiber: so-called “delicate” trichomes are present in most species, while “stout” trichomes with a swollen base are only found in Z.corallinum and Z.montanum. Oil cells occur in both epidermal layers of some species in sects. Zingiber and Cryptanthium, but only in the abaxial epidermis of Z.ellipticum in sect. Pleuranthesis. Crystals are found in the abaxial epidermis only in all species, but are present in both epidermal layers of Z.corallinum and Z.montanum. Although the epidermal morphology is similar in most Zingiber species, stomatal density, type of trichome and distribution of oil cells and crystals offer valuable information for the systematic and taxonomic studies in this genus.


Introduction
The type genus Zingiber of Zingiberaceae was established by Miller in 1754 and contains about 150 species, widely distributed from tropical to subtropical Asia (Theilade 1999;Theerakulpisut et al. 2012). The center of Zingiber diversity is located in Southeast Asia, where China has 42 species (Wu and Larsen 2000). Given the recent discovery of several new species, this figure is probably an underestimate (Bai et al. 2015a(Bai et al. , 2016(Bai et al. , 2018Li et al. 2020a;Wang et al. 2020). Zingiber has been confirmed to be monophyletic by molecular analyses (Kress et al. 2002;Li et al. 2020b) and is easily distinguished from other genera of Zingiberaceae by specific features of the flower structure; for example, the lateral staminodes are fused to the labellum; there is an elongated, horn-shaped anther crest wrapped around the upper part of style; and there is a pulvinus at the base of the petiole (Wu and Larsen 2000;Bai et al. 2015b). The traditional infrageneric classification of Zingiber recognizes four sections based on the habit and position of the inflorescence (Schumann 1904), namely sects. Zingiber (basal with long peduncle), Cryptanthium (radical with short procumbent peduncle), Pleuranthesis (spikes breaking through the leaf sheaths laterally) and Dymczewiczia (terminal inflorescence). However, some species (such as Z. junceum and Z. barbatum) have been found to have both basal and terminal inflorescences, making it difficult to place them unequivocally in a particular section (Kishor and Leong-Škorničková 2013). A recent study proposed that sect. Dymczewiczia should be merged with sect. Zingiber because palynological evidence suggests that the pollen is very similar in both sections, being spherical with cerebroid sculpturing, which differs from the ellipsoidal pollen grains with spira-striate sculpturing found in sect. Cryptanthium (Theilade et al. 1993). Phylogenetic evidence also shows that sect. Dymczewiczia is nested within sect. Zingiber in the phylogenetic tree (Theerakulpisut et al. 2012). In addition, the taxonomic identification of this genus tends to rely on floral characters, mainly flower color and labellum shape (Wu and Larsen 2000), while leaf features have received little attention. However, the flower characters are ineffective in identifying related species due to variation within the same species (Bai et al. 2015b). For example, Z. monglaense and Z. flavomaculosum have the same leaf and flower characteristics, and are considered to be merged (Tong 1998). The same was observed for Z. nudicarpum, Z. peninsulare and Z. newmanii (Bai et al. 2019). Moreover, the flower characters can be difficult to define precisely in Zingiber species, because the florescence time is short and the characters are often not well preserved in herbarium specimens (Bai et al. 2015b). Thus, more experimental studies, for example, involving leaf characteristics and leaf epidermal micromorphology, are needed on the infrageneric systematic classification of Zingiber. Leaves have some advantages over flowers, as they can be obtained at all stages of plant growth and leaf features are easier to preserve.
Leaf epidermal micromorphology, which describes the shape of epidermal cells, the outline of anticlinal walls, stomatal type, surface ornamentation and trichome type, has become a tool for the study of phylogeny and taxonomy in many plant species (Baranova 1972;Wilkinson 1979;Stace 1984), especially in those families where identification is complicated, such as Salicaceae (Chen et al. 2008;Ghahremaninejad et al. 2012;Wang et al. 2012), Rosaceae (Tahir and Rajput 2009;Zamani et al. 2017) and Lamiaceae (Moon et al. 2009(Moon et al. , 2010Eiji and Salmaki 2016;Mannethody and Purayidathkandy 2018;Gul et al. 2019). For example, papilla patterns were useful in the discrimination of several Poaceae species (Zhang et al. 2014). The type of trichome, and the form of the trichome base and anticlinal cell walls have all been valuable for the identification of Fagaceae species (Zhou and Xia 2012;Deng et al. 2013Deng et al. , 2014. The trichome type and stomatal length were instrumental in assessing the phylogeny of the Ranunculaceae (Hoot 1991;Shi and Li 2003). Based on evidence that the range of variation in leaf epidermis overlaps completely between the two families, den Hartog and Baas (1978) transferred Hippocrateaceae into Celastraceae. In Zingiberaceae, leaf anatomical characters have also proved useful for taxonomic studies. The subfamily Costoideae (= Costaceae) is anatomically very distinct from the remainder of the family, and thus supports the separation of Costoideae from the Zingiberaceae (Tomlinson 1956). The type of silica inclusion serves to distinguish the tribes Globbeae, Hedychieae and Alpinieae (Tomlinson 1956).
The leaf epidermal features of some genera of Zingiberaceae, such as Amomum, Alpinia, Boesenbergia, Kaempferia, Curcuma Hedychium, Elettaria and Globba, have been described to some extent (Tomlinson 1956;Patel 1975;Hussin et al. 2000Hussin et al. , 2001Xiao et al. 2004;Talip et al. 2005;Jayasree 2007;Chen and Xia 2010;Martins et al. 2010;Tang et al. 2010;Kajornjit et al. 2018;Salasiah and Meekiong 2018). Nevertheless, only a few representative species in these genera have been studied. So far, studies of leaf epidermis of Zingiber have only covered seven species (Olatunji 1980;Nyawuame and Gill 1990;Jayasree 2007), including two widely cultivated species (Z. officinale and Z. montanum), one widely distributed species (Z. zerumbet), and four taxa from South India, but these accounts were not detailed. All seven species studied are from two sections, sects. Zingiber and Cryptanthium (only one species, Z. wightianum), which is insufficient to represent the entire genus. In this study, we used multiple samples from the three sections of Zingiber (sects. Zingiber, Cryptanthium and Pleuranthesis) in China to investigate leaf epidermal micromorphology by light microscopy (LM) and scanning electron microscopy (SEM), and then compared the leaf epidermal characters at different classification levels in Zingiberaceae. Thus, the aim of the present study was to describe the leaf epidermal features of Zingiber and to assess their phylogenetic and taxonomic significance.

Materials and methods
More than 300 samples from 22 Zingiber species (Table 1) in China were used in the study. Leaf material from mature plants was collected by the authors in the field and voucher specimens were deposited in the herbarium of South China Normal University (SN). Fresh leaves were fixed in 90% ethyl alcohol solution, 5% formaldehyde and 5% acetic acid at a ratio of 18:1:1, and subsequently epidermal tissue was obtained from the leaves by gently scraping it off with a stainless steel blade. Pieces of leaf epidermis were stained in a solution of 1% safranin in 50% ethanol, and then dehydrated in an ethanol series before being mounted in glycerine gel for light microscopy. To ensure consistency of epidermal structure, at least five slides were examined for each sample. Twenty epidermal cells and stomata from each sample were measured and a mean was calculated based on the range of variation. The stomatal index and stomatal density were obtained for an area of 0.5 mm × 0.6 mm using the following formulae: stomatal index = number of stomatal apparatuses/ (number of stomatal apparatuses + number of epidermal cells); stomatal density = number of stomatal apparatuses per mm 2 leaf area. All statistical analyses were performed using SPSS11.5 and Microsoft Excel 2010. A confidence level of p ≤ 0.05 was considered to be significant. Material for scanning electron microscopy was macerated in 4% glutaric dialdehyde solution for about 24 h and dehydrated in a graded alcohol series, and then mounted on stubs. After gold sputtering, the specimens were examined and photographed under a JEOL JSM-6360LV scanning electron microscope. The terminologies of the stomatal complex types used in this study are those of Fryns-Claessens and Van Cotthem (1973) and Dilcher (1974).

A comparison of leaf epidermal characteristics in 22
Zingiber species is shown in Table 1.

Epidermal cells
When examined by LM, the epidermal cells of Zingiber species were found to be mostly hexagonal or polygonal, with the long axis usually perpendicular to the veins, and arranged in rows parallel to the veins; the anticlinal walls were straight to slightly curved ( Fig. 1A-F). Adaxial epidermal cells were always more regularly arranged and slightly larger than abaxial epidermal cells ( and also were usually elongated with the length 1.2-2.0 times longer than the width. The epidermal cells above the veins were smaller and more or less longitudinally elongated (Figs 1E, F, 2C). When examined by SEM, the cells were convex on the adaxial side of the epidermis ( Fig. 2A) and concave on the abaxial side ( Fig. 2B) with smooth cuticular membranes. The anticlinal cell walls were invisible or obscure.

Stomatal apparatus
The stomatal apparatus, which occurs in both the adaxial and abaxial leaf epidermis in all Zingiber species studied, was of the tetracytic type with four subsidiary cells around the stoma, one on each side and one at each pole ( Fig. 1G-L). The guard cells were reniform with smooth cuticular membranes (Fig. 2D). The lateral subsidiary cells were subtriangular with the long axis parallel to the stoma, while the terminal subsidiary cells were adjacent to the stoma poles. The stomatal orientation (the long axis of all stomata) was approximately parallel to the veins. The stomatal size in both leaf epidermal layers was similar in all species (Table 1) in the adaxial epidermis and from 36.20±0.52 × 19.86±0.21 um (in Z. zerumbet) to 49.11±1.80 × 27.23±1.66 um (in Z. leptorrhizum) in the abaxial epidermis. Stomata occurred much more frequently in the abaxial epidermis than in the adaxial epidermis in all species studied (Table 1), and most of them were randomly distributed in the intercostal regions (Fig. 1E, F). The stomatal index of the adaxial epidermis and the abaxial epidermis ranged from 0.16%±0.26% (in Z. ellipticum) to 2.87%±0.90% (in Z. montanum) and from 3.67%±0.95% (in Z. ellipticum) to 9.34%±0.58% (in

Trichomes
Trichomes were found on the abaxial surface in all species studied (Figs 2E, F, 3A-C), and occasionally also on the adaxial surface in Z. ellipticum, Z. xishuangbannaense and Z. bisectum. Two types of trichome were recorded: Type 1, a delicate, simple unicellular trichome, straight or curly, and easily detached (Figs 2E, 3A, B, D, E), which was found in all species studied except for Z. corallinum and Z. montanum; Type 2, a stout, simple unicellular trichome, straight with pointed apex and swollen trichome base (Figs 2F, 3C, F), which was found in only two species, Z. corallinum and Z. montanum.

Oil cells
Oil cells occurred frequently on the abaxial surface in all samples of the Zingiber species studied, and also occurred infrequently in the adaxial epidermis of Z. densissimum, Z. longiligulatum, Z. roseum and Z. xishuangbannaense. The oil cells were subrotund and of small size, and usually contained yellow or translucent oil droplets (Fig. 3G-I), which were easily distinguishable from epidermal parenchyma cells.

Crystals
Crystals were found in the epidermis of all Zingiber species studied. There were many crystals in the epidermis of Z. corallinum, Z. montanum, Z. longiglande, Z. tuanjuum, Z. cochleariforme, Z. guangxiense and Z. teres, but few in Z. atrorubens, Z. recurvatum and Z. leptorrhizum; crystals were rare in the remaining species. The crystals were usually rhombic and square ( Fig. 3J-L), and were mainly distributed above or near the veins in the abaxial epidermis (Fig. 3L), as well as in the intercostal regions (Fig. 3J, K), while only being present in the adaxial epidermis of Z. corallinum and Z. montanum.

Discussion
Our results show that the epidermal cells of Zingiber species are very similar in shape, i.e. hexagonal or polygonal, with non-sinuous anticlinal walls; the cells are arranged parallel to leaf veins. The results are consistent with those of previous studies on seven species of Zingiber (Olatunji 1980;Nyawuame and Gill 1990;Jayasree 2007) and other genus species in Zingiberaceae (Table 2).
Similarly to a number of other Zingiberaceae genera, the stomata of Zingiber are amphistomatic, tetracytic and aligned in a linear-axial orientation ( Table 2). The distribution of the stomatal apparatus can be useful for taxonomic studies of Zingiberaceae. For example, the stomatal apparatus of three genera, Alpinia, Elettaria and Globba, are distributed near the veins in rows, as well as being randomly distributed in the intercostal regions, while in most other genera of Zingiberaceae the stomata are always randomly distributed in the intercostal regions. These three genera can therefore be distinguished on this basis from other genera of Zingiberaceae. In addition, our results show that the stomatal density and stomatal index of the species in sect. Pleuranthesis are significantly lower than in sects. Zingiber and Cryptanthium. This suggests that stomatal density (or stomatal index) could allow species of sect. Pleuranthesis to be distinguished from other species of Zingiber.
Our results also show these two types of trichomes on the epidermis of the Zingiber species studied. Similarly to other genera of Zingiberaceae, all trichomes on the leaf surfaces of Zingiber were unicellular (Table 2). However, delicate trichomes were found on the epidermis of most species of Zingiber, while stout trichomes ("Borste") were only found in two species, Z. corallinum and Z. montanum. This indicates that Z. corallinum and Z. montanum are closely related and markedly different from other species of Zingiber. In addition, the trichomes on the epidermis of three genera (Alpinia, Amomum and Elettaria) from the subfamily Alpinioideae are all "Borste" (Table 2), while those of the genera in the subfamily Zingiberoideae are either "Borste" and/or "Weichhaare". This indicates that the type of trichome can have taxonomic significance in Zingiberaceae. According to various molecular phylogenetic trees (Kress et al. 2002, Williams et al. 2004, Liang et al. 2020, the stout trichome exists in both the derived groups (such as Kaempferia, Hedychium and Zingiber) and basal groups (such as Aplinia, Amomum and Elettaria) of Zingiberaceae, while the delicate trichome is only present in the derived groups. Within the tribe Zingibereae, the genus Curcuma only has the stout trichome, while the other three genera, Kaempferia, Hedychium and Zingiber, have both types. This suggests that the latter three genera should be closely related to each other, and more distantly related to Curcuma, consistent with the molecular phylogenetic trees (Kress et al. 2002;Liang et al. 2020). These results also suggest that the characteristics of leaf epidermal trichomes have systematic and taxonomic significance for Zingiberaceae.
Previous studies have shown that oil cells, which often occur in the mesophyll, root and rhizome (Sherlija et al. 1998, Tang et al. 2010, Uma and Muthukumar 2014, are present in all species of Zingiberaceae (Tomlinson 1959). Oil cells are responsible for the production of volatile compounds that provide fragrance for the leaves of Zingiberaceae species (Victório et al. 2011). We found that oil cells occur in the leaf abaxial epidermis of all Zingiber species, and also occur in the adaxial epidermis of three species from sect. Cryptanthium, Z. longiligulatum, Z. roseum and Z. densissimum, and one species from sect. Zingiber, Z. xishuangbannaense, but not at all in the leaf adaxial epidermis of the primitive sect. Pleuranthesis. Similarly, oil cells in most genera of Zingiberaceae are found mainly in the abaxial epidermis, but in the adaxial epidermis of only three genera of the subfamily Zingiberoideae (Table 2), Kaempferia, Globba and Zingiber. Thus, the distribution of oil cells is a useful characteristic in identifying species and sections of Zingiber, as well as genera of Zingiberaceae. Crystals are usually rhombohedral, rod-like or acicular, sometimes occurring in clusters that resemble a coarse sand, and are commonly found in the hypodermis of the lamina in families of Zingiberales, such as Musaceae, Cannaceae and Heliconiaceae, but rarely in leaf epidermis (Tomlinson 1959, 1961, Triplett and Kirchoff 1991. Crystals were recently found in costal epidermal cells on both leaf surfaces in some genera of Zingiberaceae, such as Globba, Alpinia, Amomum and Elettaria (Tomlinson 1956;Hussin et al. 2000;Talip et al. 2005;Jayasree 2007;Kajornjit et al. 2018;Salasiah and Meekiong 2018). We also found crystals in the epidermal cells of all species of Zingiber. The crystals only occur in the abaxial epidermis of most species of Zingiber, but in both epidermal layers of Z. corallinum and Z. montanum, showing that the two species Z. corallinum and Z. montanum are closely related and markedly different from other species of Zingiber. Thus, crystals in leaf epidermis can also have systematic and taxonomic significance for Zingiber.
Z. ellipticum, the sole member of sect. Pleuranthesis in China, was preliminarily identified as a new species, Plagiostachys elliptica of the genus Plagiostachys by Tong and Xia (1987) based on the character of the spike inflorescence breaking through the leaf sheaths laterally. Subsequently, it was transferred to sect. Pleuranthesis under the genus Zingiber by Wu et al. (1996), because of its spherical pollen grains and two floral characters: i) the labellum has basally connate lateral staminodes; ii) the elongated anther appendage is wrapped around the style. The above analyses of leaf epidermal micromorphology of Zingiber spp. also show that the leaf epidermal characters of Z. ellipticum from sect. Pleuranthesis are basically consistent with other Zingiber species, suggesting that this species has a close interrelationship with other Zingiber species. However, there are obvious differences in stomatal density in leaf epidermis between Z. ellipticum from sect. Pleuranthesis and the species from the other two sections of Zingiber. This suggests that the species of sect. Pleuranthesis form a distinct taxon within the genus of Zingiber. This has been confirmed by molecular phylogenetics (Kress et al. 2002;Theerakulpisut et al. 2012), but further molecular, morphological, anatomical and palynological studies are needed for a systematic classification. In addition, the two species of sect. Zingiber, Z. corallinum and Z. montanum, both of which have stout trichomes and crystals in both epidermal layers, are markedly different from other species of Zingiber. Moreover, their inflorescence and leaf morphology are similar, which can make their identification confusing. These observations highlight the need for the systematic relationship between and classification of the two species to be determined after further study.

Conclusions
As in other genera of Zingiberaceae, the epidermal cells of Zingiber are hexagonal or polygonal, with non-sinuous anticlinal walls, with the cells arranged parallel to leaf veins. Tetracytic stomata are distributed on both surfaces, and oil cells and crystals are common. Although the overall epidermal morphology is similar among Zingiber species, stomatal density, trichome type and distribution of oil cells and crystals can offer valuable systematic and taxonomic information. Two types of trichome are found in Zingiber: delicate trichomes are present in most species, while stout trichomes with a swollen base are present in Z. corallinum and Z. montanum, which is a novelty for Zingiber.