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Leaf epidermal micromorphology and pollen morphology were observed by optical microscope and scanning electron microscopy (SEM) in 10 species of C. fuma, and cluster analysis was carried out according to pollen characteristics and leaf epidermal characteristics, respectively.
It is expected to provide a necessary basis for the phylogenetic evolution and taxonomic identification of plants in this group, among which the pollen morphology of 10 species is an important investigation.
There was little difference in pollen morphology and size among the 10 plant species, which were nearly spherical, oblong spherical or oblate spherical, and the polar view was trifid nearly circular or trifid nearly triangular.
The equatorial surface is elliptical, the polar-red ratio is 0.85~1.16, and the germination pore type is three-hole type.
Micromorphological study of Camellia sinensis spp
Camellia sinensis is different from other groups of plants because of its unique morphological characteristics of "nodulous protrusions on the ovary and fruit surface", and is a group that spreads from Guizhou to surrounding provinces.
In 1939, experts discovered a specific species with nodulous protrusions in the ovary and pericarp in Sichuan Province, which was named tumor fruit tea, and experts established the tumor fruit tea group based on the unique form of "capsule peel with nodulous folds".
From only 6 species, 11 more species have been reported in the following decade. Subsequently, through the observation of specimens, the 17 species were classified into 6 species and 6 varieties.
In 1996, experts reconfirmed 15 individual species based on macroscopic morphological studies. However, due to the fact that most of the plants of the tea group grow in karst mountains and are distributed in a narrow area, field investigation is difficult.
The previous taxonomic treatment of this taxon was based on the macroscopic trait research of the specimens in the collection, and there was a lack of multidisciplinary taxonomic evidence, and some basic taxonomic problems were still left.
Therefore, supplementing the multidisciplinary data of this taxon can help to solve these taxonomic problems. As one of the evidences for plant taxonomic research, leaf epidermal micromorphology has been widely used in taxonomic research of Camellia sinensis.
Many experts analyzed the leaf anatomy, micromorphology and Fourier transform infrared spectroscopy of 18 plants in the tumor fruit tea group, and suggested that the taxonomic status of 11 species should be restored.
Although this study has made a detailed study of this group of plants, the collected plants are planted in homogeneous gardens, and lack the diversity and representativeness of wild plants.
Therefore, it is necessary to continue the study of leaf epidermis in wild populations. Moreover, pollen morphology is also one of the important evidences in taxonomic studies.
In this survey, the pollen morphology data of 10 plants in the tumor tea group were analyzed and reported, and the leaf epidermal micromorphology of 10 plants was studied.
In order to supplement the micromorphological data in the taxonomic studies of this group, and to provide reference materials for further clarifying the interspecific definition and systematics of plants in the tumor tea group.
In the field, fresh anthers are stored in glutaraldehyde and brought to the laboratory for electron microscopy scanning. Whole pollen was randomly selected for observation and photography.
ImageJ image software was used to measure the length of the polar axis and the equatorial axis. Using Excel2019 statistics, the pollen pole-red ratio was calculated.
The terms and grading standards related to pollen morphology refer to Wei Zhongxin and Wang Fuxiong, and the shape of pollen is divided into five grades according to the extreme red ratio, namely ultra-long spherical, long spherical, nearly spherical, oblate spherical and super-oblate spherical.
Pollen grain size and shape characteristics, pollenological analysis
The 10 plants in the tumor tea group were all single pollen grains, and the germination pores were three-groove and radially symmetrical, with a polar axis length of 30.86~39.37 μm, an equatorial axis length of 32.10~37.40 μm, and an extreme red ratio of 0.85~1.16.
According to the pollen morphological classification criteria, the pollen of the other eight plant species were nearly spherical, except for the oblate spherical shape of the leaf noduma fruit tea and the oblong spherical shape of the sharp bract fruit tea.
The polar plane of Liping tumor fruit tea and thick shell red tumor fruit tea was trifided regular triangle, and the polar surface of the other 8 plant species was trifid nearly round, and there were 2 kinds of pollen equatorial view, among which Liping tumor fruit tea and narrow leaf tumor fruit tea were fusiformal, and the other 8 kinds were oval.
In addition, the outer wall decoration characteristics of 10 plant species mainly showed granular, wrinkled groove or wrinkled wavy to granular forms, among which the outer wall decoration of Anlong tumor fruit tea, Leye tumor fruit tea and sharp bract tumor fruit tea was wrinkled to granular.
The outer walls of the four species of Zeng's tumor fruit tea, Liping tumor fruit tea, narrow leaf tumor fruit tea and wrinkled leaf tumor fruit tea were granular, and the other three plants of thick-shelled red tumor fruit tea, Libo red tumor fruit tea and Sanjiang tumor fruit tea were decorated with wrinkled groove pattern on the outer walls.
However, the 10 species had significant differences in the outer wall ornamentation, mesh size and morphology, which indicated that pollen ornamentation could be used as one of the main interspecific taxonomic traits of the plants of the Tumor Fruit Tea Formation and had important taxonomic value.
According to 14 structural indexes such as pollen wall decoration, equatorial view, pollen shape and size, R-type cluster analysis was carried out for 10 plant species.
At the Euclidean distance of 4.5, the 10 plants were divided into 6 categories, for example, Liping tumor fruit tea and narrow leaf tumor fruit tea were the closest, and the outer wall decoration of the pollen of these two plants had the characteristics of granular and fusiform pollen equatorial view.
These two kinds of plants form a branch together with Leye Tumor Fruit Tea and Zeng's Tumor Fruit Tea. Thick-shelled erythroma fruit tea and sharp bract fruit tea form another category.
When the Euclidean distance was 15, the 10 species of the Tumor Tea group were divided into two groups, and the first class was composed of 8 species: Liping Tumor Fruit Tea, Narrow Leaf Tumor Fruit Tea, Leye Tumor Fruit Tea, Zeng's Tumor Fruit Tea, Thick Shell Red Tumor Fruit Tea, Sharp Bud Tumor Fruit Tea, Libo Red Tumor Fruit Tea and Wrinkled Leaf Tumor Fruit Tea.
These species have the characteristics of shorter equatorial axis and longer germination groove, and the second class is composed of Anlong tumor fruit tea and Sanjiang tumor fruit tea, and the pollen morphology of the two plants is less different.
Morphological characteristics and cluster analysis of leaf epidermis
According to the observation, the morphology of leaf epidermal cells of 10 plant species is diverse, which can be roughly divided into five categories: irregular wave type: the epidermal cells are irregular and unequal in size, and the vertical periphery wall is wavy.
For example, the upper epidermal cells of Tsang's tumor tea, thick shell tumor fruit tea, Libo red tumor fruit tea and Tsang's tumor fruit tea, Thick shell red tumor fruit tea, Libo red tumor fruit tea and narrow leaf tumor fruit tea.
Irregular shallow wave type: the epidermal cells are irregularly shaped, unequal in size, and the vertical periphery wall is superficial, such as the upper epidermal cells of Libo Rhodoma fruit tea and the lower epidermal cells of Anlong tumor fruit tea, sharp bract tumor fruit tea and Leye tumor fruit tea.
Irregular deep-wave type: the epidermal cells are irregularly shaped, unequal in size, and the deep-wavy vertical periphery wall is significantly thickened, which is found in the upper epidermis of thick-shelled rubnoma fruit tea, the upper and lower epidermal cells of Sanjiang tumor fruit tea and the lower epidermis of wrinkled leaf tumor fruit tea.
Irregular straight curve, the epidermal cells are irregular, but the vertical periphery wall style is straight curved, which is relatively regular, and is only found in the upper epidermis of Anlong tumor fruit tea, Leye tumor fruit tea and Liping tumor fruit tea.
Polygonal, upper epidermis of Libo red tumor fruit tea and lower epidermis of Sanjiang tumor fruit tea. The area of leaf epidermal cells was measured by ImageJ software, and 30 cells of each plant were measured and averaged.
Among the upper epidermal cells, the smallest cross-sectional area was 1036.77 μm2, and the maximum was 1036.77 μm2, among which Anlong tumor tea was the closest to Zeng's tumor fruit tea, with the maximum cell length-to-width ratio of 1.38 and the minimum value of 1.11.
The analysis of lower epidermal cells showed that the minimum cross-sectional area of the cells was the thick-shelled rubnoma fruit tea, and the maximum cell size was the one with the narrow leaf tumor fruit tea, and the cell length to width ratio was 1.79 and the minimum was 1.19.
Among them, the aspect ratio of lower epidermal cells in Leye tumor fruit tea and narrow leaf tumor fruit tea was 1.26, and it was found that there were different numbers of glands in the epidermis of three plant species.
In addition, there were more oil glands in the lower epidermis and a small number of glands in the upper epidermis, a small number of glands in the upper epidermis of the tea and more glands in the upper and lower epidermis of Libo rubruma tea.
According to the R-type cluster analysis of 8 structural indexes of leaf epidermis, including stomatal density, stomatal index, cell shape and vertical wall style, 10 plants were divided into 6 groups when the Euclidean distance was 3.
Among them, Leye tumor fruit tea and Sanjiang tumor fruit tea are one branch, and the morphology and cross-sectional area of the upper epidermal cells of leaf epidermal cells are relatively similar in these two plants.
Therefore, Anlong tumor fruit tea, Libo red tumor fruit tea, sharp bract tumor fruit tea and Liping tumor fruit tea were one branch, indicating that they were closely related, and the other four species each formed a branch.
When the European distance was 10, the 10 species were divided into 3 groups, among which 8 species, including Anlong tumor fruit tea, Libo red tumor fruit tea, sharp bract tumor fruit tea, Liping tumor fruit tea, narrow leaf tumor fruit tea, Zeng's tumor fruit tea, Leye tumor fruit tea, and Sanjiang tumor fruit tea, were clustered into one category due to the characteristics of higher length-width ratio and similar stomatal density.
However, the thick-shelled rubhoma fruit tea is a separate category due to the small area of leaf epidermal cells and low stomatal density, while the leaf tumor fruit tea is a class because of the large area of lower epidermal cells and the oil gland point only in the upper epidermis.
The pollen morphology and leaf epidermal micromorphology of the 10 species tested in the Tumor Tea Group were different among species, indicating that these traits had certain reference value for the classification of the Tumor Fruit Tea Group.
It can be used as one of the bases for interspecific identification, and can make up for the classification problems that are difficult to solve by relying on macroscopic traits to a certain extent.
However, in the field investigation, we found that the interspecific taxonomy of plants in the Tumor Fruit Tea Group was complex, and interspecific hybridization occurred frequently.
Therefore, field surveys should be carried out on a population basis, and a multidisciplinary approach should be used to solve the taxonomic problems of this taxonomic group