Characteristics of Leaf Epidermis of 22 Lilieae (Liliaceae) under Different Altitudes in China ()
1. Introduction
Leaves are critical for plant growth and are directly exposed to the air environment. Stomata are the pores on the surface of leaves, flanked by guard cells, that regulate the gas exchange between the internal plant tissues and the atmosphere, especially water vapor and CO2 [1] [2]. In addition, stomatal characteristics have been shown to correlate with the environmental factors in their habitat. Their quantitative properties, such as stomatal size and stomatal density, are sensitive to changes in environmental factors such as temperature, CO2 concentration, precipitation and light [3]. Since there is a clear linear correlation between environmental factors and altitude, significant statistical relationships between stomatal characteristics and altitude have been documented in many species [4] [5] [6]. However, when research interest is focused on higher levels of taxonomy, such as genera, tribe, families, etc., it is not clear whether changes in stomatal characteristics between species also vary linearly with altitude.
Tribe Lilieae [7] [8], belonging to the family Liliaceae, contains four genera: Lilium L. (including Nomocharis Franch.), Fritillaria L., Notholirion Wallich ex Boissier and Cardiocrinum (Endlicher) Lindley. This tribe is the species-abundant taxonomic group with about 91 species in China (61 species in Lilium, 24 species in Fritillaria, three species in Notholirion, and three species in Cardiocrinum) [9]. Many species of this tribe have been cultivated worldwide for ornamental, and some species are used as folk medicine [10] [11]. For example, the bulbs of Fritillaria cirrhosa D. Don, F. unibracteata Hsiao et K. C. Hsia, F. przewalskii Maxim., F. delavayi Franch. are widely used as a traditional Chinese medicine named “Chuan bei mu” for the treatment of cough and chest congestion; Lilium lancifolium Thunb., L. brownii F.E. Brown and L. pumilum DC. are known as “Bai he”, which are used to soothe the nerves [10]. Numerous species of Lilium, Fritillaria, and Cardiocrinum, are commonly cultivated as potted plants or cut flowers for their bright color and fragrance [11].
Members of the tribe Lilieae grow in vastly different habitats in China. For example, the altitudinal distribution of tribe Lilieae members ranges from 0 m (L. tsingtauense Gilg, near sea level, in Shandong, China) to 5100 m (F. afusca Turrill, live in moist and gravelly places, in Southern Xizang, China) [9]. This tribe therefore represents an excellent system to study the relationship between leaf epidermis and environmental factors.
In this study, 22 Liliaceae species were collected from different localities in six provinces of China to explore whether there is a detectable altitude correlation in the dynamics of leaf stomatal characteristics. The aim of the present work is to answer a fundamental question: is the leaf stomatal affected by the elevation factor at the tribe level? In addition, the leaf epidermis characteristics, which currently contains the entire Tribe Lilieae, are poorly studied, thus this data also contributes to the taxonomy at the tribe level.
2. Materials and Methods
2.1. Plant Material
A total of 22 taxa belonging to the tribe Lilieae were studied (Table 1). Materials have been collected at different localities in the field except F. anhuiensis S. C. Chen & S. F. Yin (cultivated material). Plant samples were made into herbarium specimens, from which leaf study material was obtained. The vouchers of all the collections were deposited in the herbarium of Kunming University.
2.2. Indoor Sample Treatment
For leaf epidermis examination, mature, fully expanded leaf samples were obtained
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Table 1. Spatial distribution of tribe Lilieae samples.
from herbarium specimens. The Sample treatment includes four steps according to the method of Wang et al. [12]: 1) leaf blades were selected from each specimen, cut into 1 × 1 cm2 pieces, and place it into a 10 ml centrifuge tube; 2) after the addition of glacial acetic acid and 30% H2O2, the mixture was heated at 60˚C for 6 h in a water bath; 3) take out the samples and wash them clean, then separate the adaxial and abaxial epidermis under the stereomicroscope; 4) the separated epidermis were dyed using 1% sarranine solution to make section with glycerin to mounted and then sealed with nail polish.
2.3. Microscopic Image and Data Acquistion
Include two steps: 1) microsope images were acquired with the LUMIX LX3-GK attached to a ZEISS Axioskop 20 microsope; 2) collect image data on computer using Adobe photoshop CC 2018 and ImageTool 2.0 software.
2.4. Measurement of Stomatal Traits
For the present analyzes, two indices [stomatal area (SA) and stomatal index (SI)] were utilized to describe stomatal quantitative characters. Stomatal shape is elliptic in all species examined. Therefore, their areas (in Table 2) can be conveniently calculated from the area formula of the ellipses. SI values were computed as in
, (1)
where S = number of stomata per unit area, and E = number of epidermal cells per same unit area. Stomatal terminology was based on that proposed by Dilcher [13], while the classification of pavement cells was based on the work of Wang and Tao [14].
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Table 2. Summary of selected leaf epidermal features in tribe Lilieae taxa.
2.5. Statistical Analysis
Analysis of variance (ANOVA) followed by the least significant difference test (LSD) was performed on stomatal traits to indicate any significant difference among the taxa studied. The correlations of two stomatal indices (SA and SI) and altitude were disposed of by Bivariate Correlations Analysis. Pearson’s coefficient of correlation was determined to assess the correlations between the different indices. Only correlations significant at the 1% level or higher are discussed. Statistical and plot analyzes were performed using the software SPSS 27 and Origin 2022, respectively.
3. Results
A summary of the micromorphological characteristics of the leaf epidermis is given in Table 2, and depicted in Figure 1, and Figure 2. The pavement cell properties of 22 species were described. Two stomatal indices, SA and SI, for 22 species were used for linear regression with increasing altitude, respectively.
3.1. Qualitative Character of Leaf Epidermis
The pavement cells of the tribe Lilieae as seen under light microscope are linear, polygonal or irregular in form (Figure 1, Figure 2, and Table 2). It is interesting that the shape of cells in the upper and lower epidermis in most taxa is uniform except five taxa. These taxa are Fritillaria tortifolia, Lilium bakerianum var. delavayi, L. nanum var. nanum, L. sulphureum, L. wenshanense, and L. sealyi. In general, both in Notholirion and Fritillaria, the shape of the pavement cells is linearly specific, and in Cardiocrinum the epidermal cells are irregular in shape. While Lilium taxa possessed both polygonal or irregular form, it is dependent on species.
The pattern of anticlinal walls separates the 22 taxa into two groups. Group one comprises genus Fritillaria, Notholirion, and some taxa of Lilium, namely L. bakerianum var. delavayi, L. nanum var. nanum, and L. sulphureum, have straight anticlinal cell walls (Figure 1, Figure 2, and Table 2), while the second group comprising genus Cardiocrinum and the rest taxa of Lilium, have microsinuous to deep sinuous anticlinal cell walls (Figure 1, Figure 2, and Table 2).
Only one species, namely F. tortifolia, possessed trichomes on the abaxial surfaces (Figure 2), while the other species lacked epidermal hairs. Additionally, this species is hypostomatic (stomata on the abaxial surface only).
3.2. Quantitative Character of SA and SI
ANOVA indicated that the differences between SI of different genus were indistinctive, except Lilium and Fritillaria (p = 0.048). As for SA, there is not any distinction between Lilium and Cardiocrinum, Lilium and Fritillaria, Cardiocrinum and Fritillaria. However, the “p-value” of the difference between Fritillaria and Notholirion is 0.008, which is significant. While the difference between Lilium and Notholirion (p = 0.040), Cardiocrinum and Notholirion (p = 0.038) is least significant.
3.3. Relationship between Stomatal Traits and Altitude
As the leaf epidermis traits were different between taxa, we analyzed the relationships between the leaf epidermis traits and altitude separately. SA increased with elevation. There was a good positive correlation between altitude and SA
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Figure 1. Micromorphological characteristics of the adaxial leaf surfaces. (A) Cardiocrinum cathayanum; (B) C. giganteum var. yunnanense; (C) Fritillaria anhuiensis; (D) F. crassicaulis; (E) F. tortifolia; (F) Lilium apertum; (G) L. bakerianum var. delavayi; (H) L. brownii; (I) L. duchartrei; (J) L. leucanthum; (K) L. meleagrinum; (L) L. nanum; (M) L. pardanthinum; (N) L. primulinum var. burmanicum; (O) L. primulinum var. ochraceum; (P) L. sealyi; (Q) L. sulphureum; (R) L. taliense; (S) L. tsingtauense; (T) L. wenshanense; (U) Notholirion bulbuliferum; (V) N. campanulatum. Scale bars = 100 μm.
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Figure 2. Micromorphological characteristics of the abaxial leaf surfaces. (A) Cardiocrinum cathayanum; (B) C. giganteum var. yunnanense; (C) Fritillaria anhuiensis; (D) F. crassicaulis; (E) F. tortifolia; (F) Lilium apertum; (G) L. bakerianum var. delavayi; (H) L. brownii; (I) L. duchartrei; (J) L. leucanthum; (K) L. meleagrinum; (L) L. nanum; (M) L. pardanthinum; (N) L. primulinum var. burmanicum; (O) L. primulinum var. ochraceum; (P) L. sealyi; (Q) L. sulphureum; (R) L. taliense; (S) L. tsingtauense; (T) L. wenshanense; (U) Notholirion bulbuliferum; (V) N. campanulatum. Scale bars = 100 μm.
across Lilieae (r2 = 0.294, p = 0.009, Figure 3(A)), indicating that increases in altitude were generally accompanied by increasing stomatal area.
SI tended to decrease with increasing elevation. SI was inversely related to the altitude (r2 = −0.254, p = 0.017, Figure 3(B)), indicating that increases in altitude were generally accompanied by decreasing stomatal number.
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Figure 3. The relationships between stomatal traits and altitude.
4. Discussion
Taxonomic Implications of Leaf Epidermal Features
Given that the quantitative stomatal characters play only a minor role in delimiting genera, it is plausible to assume that they seem influenced mainly by environmental rather than genetic. For example, ANOVA shows that SI can distinguish only Lilium and Fritillaria (p = 0.048). Similarly, SA was slightly useful to delimit generic circumscriptions between Fritillaria and Notholirion (p = 0.008), Lilium and Notholirion (p = 0.040), Cardiocrinum and Notholirion (p = 0.038).
Contrary to the quantitative stomatal characters, the shape of the epidermal cells and the pattern of the anticlinal walls provide some useful taxonomic information to distinguish the genus. For example, C. cathayanum and C. giganteum var. yunnanense are very close to each other in phylogenetics but live in the different habitat characteristics (Table 1). However, there are few differences between these two taxa for epidermis character (Table 1, Table 2; Figure 2(A), Figure 2(B); Figure 3(A), Figure 3(B)). An equivalent situation has been found in L. primulinum var. burmanicum and L. primulinum var. ochraceum (Table 1, Table 2; Figure 2(L), Figure 2(M); Figure 3(N), Figure 3(O)).
In general, both in Notholirion and Fritillaria, the shape of the epidermal cells is linearly specific, while Lilium taxa possess either a polygonal or irregular form. The morphogenesis of lobed plant cells has been considered to controlled by microtubule and/or actin filament organization [15], indicating the shape of epidermis is correlated with genetic factors.
Some variation has been found between different species, and it has been observed that each species has its own unique combination of features that set them apart from each other. As a result, leaf epidermal features provide some taxonomic information at the species level. However, none of the stable traits are unique to a genus. Consequently, the epidermal characters of the leaves can only play a very minor role in defining the genus.
Among leaf morphological characters, trichomes are one of the most important traits contributing to plant’s passive resistance to pathogens, pests and drought [16]. In the sampled species, only F. tortifolia, possessed trichomes on the abaxial surfaces (Figure 3). In fact, the species was collected from the wild in Yumin County, Xinjiang, and without being stressed by pests and diseases. Xinjiang is one of China’s major drought-prone regions, especially in Yumin County. The annual average rainfall in Yumin County is 304.1 mm [17], which is far less than the other location of the present study.
Stomatal variability is mainly caused by environmental factors such as radiation, humidity, temperature, etc. [3]. As altitude increases, environmental factors increase or decrease [18]. However, the environmental factors in this study do not change linearly, but temperature. Theoretically, as temperature drops, water stress exacerbates. The drop in temperature may lead to physiological drought of plants [12]. Plants respond to drought stress by activating hormonal and genetic mechanisms that reduce the number of stomata [19]. This may be the reason for the negative correlation between SI and altitude changes in the present study (r2 = −0.254, p = 0.017, Figure 3(B)).
As air temperature continuously drops and plant physiological drought strengthens, water conservation to resist drought becomes the first essential problem to maintain the survival of plants [20]. Through regulating stomatal development and opening-closing movement, plants adapt to the environment and compensate for the insufficient water caused by low-temperature physiological drought [12]. In this study, an analysis of the relationship between altitude and SA showed these two parameters were positively correlated (r2 = 0.294, p = 0.009, Figure 3(A)), indicating that increases in altitude were generally accompanied by increasing stomatal area. Generally, there is a clear negative relationship between the size of the stomatal pore and sensitivity to increasing drought [21]. As a result, species at higher altitudes may become less sensitive to physiological drought through stomatal enlargement.
Stomatal traits are interrelated and act together and should not be viewed in isolation. Plants regulate the allocation of energy within the plant through a trade-off between traits to improve plant adaptability. Therefore, the adaptation of plants to the environment is the common adaptation of multiple traits [4].
5. Conclusions
Thus, the pattern of leaf epidermal cells and anticlinal walls provides some useful taxonomic information to distinguish species. However, none of the stable traits are exclusive to a genus. Consequently, the epidermal characters of the leaves can only play a very minor role in distinguishing the genus in tribe Lilieae.
The results of the present study confirmed that variations in the SA and SI of 22 species belong to tribe Lilieae at different locations showed a clear correlation with altitude. From a plant physiological point of view, this linear change in stomata may be related to adaptation to the physiological drought induced by temperature drops.
As a result, the pattern of leaf cells and anticlinal walls is influenced by genetic factors, while the stomatal area and stomatal index are influenced by environmental factors. Members of the tribe Lilieae have a relatively stable elevation range, which is related to their long-term adaptation to the local environment in the structure of their leaf epidermis.
Acknowledgements
We thank Mingzhong Mo (Forestry and Grassland Bureau of Honghe Hani and Yi Autonomous Prefecture, Yunnan, China) for his assistance with field sampling. This work was supported by the National Natural Science Foundation of China [Grant No.31860107].