Waterlily (Nymphaea L.) is a perennial aquatic plant, with more than 50 native species and more than 1 000 cultivated varieties (Huang et al., 2008, China Forestry Press, pp.23-28; Li et al., 2019). According to its demand for survival temperature, it can be divided into two ecological types: cold tolerant Nymphaea species and tropical Nymphaea species. The former is mainly naturally distributed in subtropical and temperate regions, and the latter is mainly distributed in tropical regions. The flower colors of tropical Nymphaea species are more abundant than cold tolerant species, but cold tolerance is poor, and it is impossible to overwinter naturally in northern regions, which is the main limiting factor affecting the introduction and cultivation of tropical Nymphaea species in northern regions.

Temperature is an important factor affecting the growth of plants. Plants form an adaptive anatomic structure in the long-term adaptation to specific environmental conditions. In the evaluation of cold tolerance of plants, structural characteristics are also an important reference index. In which, as one of the main exposed organs of plants, the anatomical structure of leaves not only affects the physiological and biochemical functions of plants, but also reflects the adaptability of plants to environmental temperature. The results showed that the thickness of upper epidermis, the thickness of spongy tissue, the tissue structure tightness and the stomatal density of Camellia oleifera leaves were the main indicators affecting the cold resistance (Zeng et al., 2020), while the thickness of palisade tissue, the palisade tissue-spongy tissue ratio and the thickness of main vein were the main indicators affecting the cold resistance of Michelia plants (Li et al., 2017). As an important index, the morphological and anatomical characteristics of leaves have been applied in the evaluation of the cold resistance of Hemerocallis fulva (Di et al., 2019), Caragana sinica (Ma et al., 2020), Cinnamomum camphora (Wang et al., 2013) and other plants. The evaluation indexes are different among different plants, but in general, the closer the leaf structure is, the stronger the cold resistance is. At present, the research on Nymphaea mainly focuses on genetic diversity (Su et al., 2020), cold and shade tolerance of varieties (Ji, 2016; Liu et al., 2020), chemical component extraction (Huang et al., 2020), and other fields. The anatomy of the leaves of Nymphaea is mainly aimed at cold tolerant species (Chen et al., 1987; Hu et al., 2003), and the study on the anatomical structures of the other four subgenera of Nymphaea has not been reported. In this study, one kind of cold tolerant Nymphaea and five kinds of tropical Nymphaea were taken as the research objects. The anatomical structure of their leaves was observed to evaluate the difference of their cold tolerant ability, so as to provide a basis for the promotion, application and new variety cultivation of Nymphaea.

1 Results and Analysis

1.1 Comparison of leaf thickness of different Nymphaea

The average thickness of the leaves of the six Nymphaea species is 427.90 μm (Table 1). There are significant differences among different species, among which Nymphaea alba has the thickest leaves, which is 803.66 μm. The leaves of Nymphaea potamophila are the thinnest (145.50 μm). Thickness is an important index to measure the cold resistance of plant leaves. The thicker the leaves, the higher the water content. Under low temperature conditions, it can delay the damage to the leaves. Among the six Nymphaea species, cold resistant Nymphaea alba have the thickest leaves, which are significantly higher than the other four subgenera. It is also consistent with the daily observation results.

1.2 Characteristics of leaf epidermis of different Nymphaea

The upper and lower epidermis of Nymphaea leaves are composed of monolayer cells, and the thickness of the lower epidermis is 12.55~22.35 μm. N.minuta is the highest, which is significantly higher than the other five species. The thickness of the upper epidermis of Nymphaea leaves is slightly lower than that of the lower epidermis, with a thickness of 7.64~16.79 μm. And the N.immutabilis is the highest, and the thickness of the upper and lower epidermis of the leaves of N. potamophila is lower than that of the other five Nymphaea species (Figure 1).

The stomata of different Nymphaea are long oval to round, which are only distributed on the upper epidermis of leaves. The average size of stomata is 130.08 μm², the average density is 548.35 /mm², and the coefficient of variation of stomatal density among different Nymphaea is 22.82%. The stomatal density of N.alba is the lowest (358.33 /mm²). The stomatal density of two species of Subgen. Brachyceras Casp. is close, higher than that of the other four subgenera. The coefficient of variation of stomatal area among different Nymphaea was 22.47%. The stomatal area of N. lotus was the largest, which was 152.69 m², and that of N.immutabilis was the smallest of 80.27 μm² (Figure 2).

1.3 Structural characteristics of mesophyll in different Nymphaea species

The mesophyll structure of Nymphaea leaves is divided into palisade tissue and sponge tissue. Palisade tissue is composed of relatively closely arranged cylindrical cells, with a thickness of 33.74~145.30 μm. Compared with Michelia, Cinnamomum camphora and other plants, its structure is loose, which may be due to its participation in the ventilation process of upper epidermal pores. While the sponge tissue is composed of irregular cells, with a thickness of 99.91~625.16 μm (Figure 2). Among the six kinds of Nymphaea, the thickness of palisade tissue and sponge tissue of N.alba is the largest, and that of N. potamophila is the smallest, which is significantly different from other Nymphaea. N. lotus and N. potamophila have higher palisade tissue-spongy tissue ratio and tissue tightness, while N.alba have lower palisade tissue-spongy tissue ratio and tissue tightness than other Nymphaea, which are 0.26 and 18.64, respectively. The tissue structure looseness of the six Nymphaea was 63.05~77.78, in which N.alba was the highest, and N. potamophila was the lowest. In general, the leaf structure of N. potamophila is the most compact among the six Nymphaea species, while N.alba is relatively loose.

1.4 Comprehensive evaluation of cold resistance of different Nymphaea species

1.4.1 Cluster analysis and screening of cold resistance related indicators

After the standardization of 10 anatomical structure indexes of Nymphaea leaves, cluster analysis was carried out. The 10 indexes could be clustered into 3 groups. Group Iincluded 5 indexes: total thickness of leaves, thickness of palisade tissue and sponge tissue, tissue looseness, and thickness of upper and lower epidermis; Group IIonly included stomatal density. Group IIIincluded the palisade tissue-spongy tissue ratio, tissue tightness and stomatal size (Figure 3).

The correlation matrix of leaf structure indicators is shown (Table 2), and the typical indicators of the three types of indicators are determined according to the correlation index calculated by formula (5). The results showed (Table 3) that the typical indicators of the three types of indicators are total leaf thickness, stomatal density and palisade tissue-spongy tissue ratio.

1.4.2 Comprehensive evaluation results

According to formulas (6) and (7), calculate the membership function values of the typical indicators of each Nymphaea, and then obtain the average membership degree of its cold resistance. The larger the average membership degree is, the stronger the cold resistance of the Nymphaea leaves is. The results showed that the cold resistance of the six Nymphaea species leaves is as follows: N.alba>N. potamophila>N. lotus>N.immutabilis>N.colorata>N.minuta (Table 4).

2 Discussion

As the main channel for gas exchange and transpiration of plant leaves, the differences in stomatal size and density can reflect the strength of plants' adaptability to adversity (Hee-Myong et al., 2001). It is generally believed (Fei et al., 1999; Wang et al., 2016) that plants with small stomatal density and stomatal size have relatively low respiratory efficiency and less water loss, so they have strong cold resistance. However, some studies (Wang et al., 2013; Zeng et al., 2020) have shown that there is little correlation between stomatal density, stomatal size and cold resistance. In this study, the high stomatal density of the tropical waterlily may be due to its distribution in the tropics, which is more conducive to transpiration and heat dissipation in the summer with high temperature and high light, while the cold resistant Nymphaea alba is on the contrary. The actual observation shows that the tropical waterlily has less leaf burns in summer, but the leaves are more prone to frostbite in late autumn. Overall, a small stomatal density may be more conducive to the cold resistance.

Many studies have shown that mesophyll tissue is closely related to the cold tolerance of leaves, but the results of different plants are different. Experiments showed that rose and camphor trees with strong cold resistance have thick palisade tissue, while sponge tissue is relatively thin. However, some studies have shown that the increase of palisade tissue, spongy tissue and leaf thickness is the result of plant adaptation to low temperature environment (Cai and Song, 2001). The results of this experiment showed that the thickness of leaves, palisade tissue and sponge tissue of Nymphaea alba were significantly higher than that of tropical Nymphaea, which was consistent with the evaluation results of cold tolerance. However, the palisade tissue-spongy tissue ratio of Nymphaea alba leaves is the lowest among all Nymphaea species, which is contrary to its evaluation results. At the same time, the anatomical results of other tropical Nymphaea species leaves are not completely consistent with their cold tolerance ranking, indicating that when evaluating the cold tolerance of Nymphaea, multiple indicators should be considered comprehensively, and one index should not prevail.

In this study, 10 indexes of leaf anatomical structure were selected to measure the five subgenera Nymphaea. Using cluster analysis method, three indexes of total leaf thickness, stomatal density and palisade tissue-spongy tissue ratio were selected to evaluate the cold resistance of Nymphaea. The results showed that Nymphaea alba had the strongest cold resistance, followed by Nymphaea potamophila, Nymphaea lotus and Nymphaea immutabilis in tropical waterlilies. Among the Subgen. Brachyceras Casp., Nymphaea colorata and Nymphaea minuta have the lowest structural cold tolerance. Subsequent studies can further determine the physiological indicators of Nymphaea leaves under low temperature stress, and then obtain more accurate results.

3 Materials and Methods

3.1 Experimental materials

The tested waterlily (Nymphaea L.) was taken from the waterlily Resource Garden of Shanghai Chenshan Botanical Garden, including Nymphaea lotus, Nymphaea immutabilis, Nymphaea colorata, Nymphaea minuta, Nymphaea potamophila and Nymphaea alba. Three plants of each species with similar growth potential were selected as the research objects, and fresh and mature leaves of Nymphaea were taken for standby in May 2020 (Table 5).

3.2 Experimental methods

3.2.1 Stomatal observation

Take the parts on both sides of the main vein of waterlily leaves, cut the leaves into small squares of 5 mm, fix them with glutaraldehyde, wash them with PBS buffer, dehydrate them with ethanol gradient, and then replace them with isoamyl acetate. After drying and spraying gold, observe and take photos under the scanning electron microscope. Select 10 visual fields for each treatment, take the average value, and calculate the stomatal density. Photoshop was used to measure and calculate the stomatal size and coefficient of variation, where the stomatal size= π×stomatal length × stomatal width/4 (Gong et al., 2010).

3.2.2 Leaves structure observation

Took the central area of mature Nymphaea leaves, cut a 1 cm length segment away from the main vein, fixed, embedded and sliced it, and dyed it with safranine and solid green. Observed it with a microscope, and took photos with five fields of vision for each slice. Photoshop software was used to measure the thickness of leaves, upper epidermis, thickness of palisade tissue, thickness of spongy tissue and lower epidermis.

According to the method of Ma et al. (2020), calculated the palisade tissue-spongy tissue ratio, tissue structure tightness, tissue structure looseness and coefficient of variation of Nymphaea leaves respectively:

(1) Palisade tissue-spongy tissue ratio of leaves=Thickness of palisade tissue/Thickness of spongy tissue

(2) Tissue structure tightness=Thickness of palisade tissue/Thickness of leaves×100

(3) Tissue structure looseness=Thickness of spongy tissue/Thickness of leaves×100

(4) Coefficient of variation=Standard deviation/Mean×100%

3.2.3 Comprehensive evaluation of cold resistance

After the standardization of anatomical structure indexes of Nymphaea leaves, cluster analysis was carried out, and the relevant indexes of each index were calculated according to the method of Zeng et al. (2020): (5)T=Σr²/(n-1), where: T is the correlation index of each anatomical index in their respective classification, n is the number of indicators in each category, and r is the correlation coefficient between each index and the indicators of the same group.

The membership function method was used to evaluate the cold resistance of different Nymphaea leaves. The index that is positively correlated with the cold resistance was calculated as follows: (6)Y(X_i)=(X_i-X_min)/(X_max-X_min)

The index calculation formula negatively related to cold resistance was as follows: (7)Y(X_i)=1-(X_i-X_min)/(X_max-X_min), where: Y(X_i) represents the subordinate value of cold tolerance of Nymphaea, X_i represents the score value of the i^th factor, X_max is the maximum score value of the i^th factor, and X_min is the minimum score value of the i^th factor.

Authors’ Contribution

YK is the executor of this research design and experiment, completed data analysis and paper writing. The author read and approved the final manuscript.

Acknowledgements

This study was supported by the Science and Technology Project of Shanghai Landscaping & City Appearance Administrative Bureau (G182424; G192406).

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