Studies of the pollen characteristics and the taxonomic significance of Impatiens from the Yunnan–Guizhou Plateau

The plant materials were primarily derived from field studies and sampling, and the collected information was recorded (Annex 1). The change in height of flower colour among the species of Impatiens increases the difficulty of classifying them (Chen, 1978). In this study, we used 1, 2, 3 and higher numbers when necessary to mark Impatiens of the same species but different colours. In the same type of Impatiens, more than two types of mature pollens were collected and stored in a test tube or collection tube filled with FAA fixatives.

The pollen of 35 species of Impatiens was observed by SEM. The pollen samples were prepared by first soaking the material and then gently removing the anthers with tweezers and dissection needles. The anthers were placed in a 2 mL collection tube and dehydrated with an alcohol gradient of 30%, 45%, 60%, 75%, 90% and 95%. The solution was replaced with anhydrous alcohol twice for 1 h at a time. The anthers were then removed with tweezers and dissected using a needle, so that the pollen was evenly distributed on the sample table with conductive glue. Under the vacuum conditions of an ion sputtering instrument (Cressington Scientific Instruments, Watford, UK), the gold spray was coated for 2–3 min. It was observed by a Zeiss scanning electron microscope (Zeiss, Jena, Germany).

The pictures were analysed using Image J (NIH, Bethesda, MD, USA), and the morphological characteristics were named based on the pollen research literature (Yu et al., 2016; Mazari et al., 2017; Hu et al., 2020; Raza et al., 2020). There were slight modifications such as pillow shape, which was named so because its shape is similar to that of ancient Chinese porcelain. When the mesh density (MD) was 12 × 1 000-fold magnification, the number of mesh was regarded as the MD.

The statistical data of Microsoft Excel YEAR (Redmond, WA, USA) were used, and SPSS 26.0 (IBM, Inc., Armonk, NY, USA) was used for correlation, principal component and system clustering analyses (Pérez-Gutiérrez et al., 2015; Zafar et al., 2022). The number of sepals (NS), which is one of the important bases for the classification of Impatiens, was added as a reference index to compare the importance of pollen characteristics during the analysis of indices.

Based on the study and analysis of pollen micromorphology of 35 species of Impatiens, the data of 19 characteristics indexes were obtained, including pollen morphology (Table 1), pollen germination pore characteristics, pollen size and mesh characteristics (the data sheet is provided in Annex 2). The pollen characteristics were primarily divided into two parts, qualitative and quantitative traits.

There were six indices of qualitative characteristics (Figure 1), which included epidermis ornamentation (EO), polar view (PV), equatorial view (EV), characteristics of germination pores (CGP), mesh feature (MF) and ridge feature (RF). The characteristics under each index were different, and the proportion was also different, indicating that the characteristics of pollen qualitative indices were rich, which aided the interspecific classification of Impatiens plants (Figure 2). There were 14 quantitative characteristic indices. The NS was the reference group, and the others included the number of germination pores (NGP), germination pore length (GPL), germination pore width (GPW), germination pore length/germination pore width (L/W), MD, ridge width (RW), polar axis length (P), equatorial major axis length (E1), equatorial short axis length (E2), pollen volume (V) and P/E1, P/E2 and E1/E2. There were differences among the species, and there was an obvious difference between the maximum and minimum values (Table 2), such as volume. The maximum value was more than 14 000 μm³, the minimum value was approximately 4 000 μm³ and the average value was approximately 8 000 μm³. Based on the aforementioned analysis described, the pollen characteristics of Impatiens were rich in diversity, which can provide positive conditions to classify the members of this genus.

Figure 1.

Figure 2.

There was some correlation among the pollen characteristics (Figure 3). Among the 19 traits of pollen, there were significant correlations among some indices, such as the SN and polar morphology, equatorial morphology, number of germination holes, MD, RW, polar axis length, equatorial minor axis length, P/E1, P/E2 and E1/E2. With reference to the NS, there was some correlation between pollen characteristics, and there was also some correlation with the NS. I. guizhouensis and I. auriculata were used as examples to show that the correlation was significant for plant classification. In the classification index, there was some relationship between the index and the index. The correlation showed that the pollen characteristics of Impatiens plants may be accompanied, and as the basis of plant identification, pollen characteristics can promote the acquisition of species information.

Figure 3.

Based on the quantitative data analysis between groups of Impatiens (Figure 5; the data sheet is shown in Annex 3), no significant differences in the quantitative index of germination pore between groups were identified, which further verified that the quantitative index of the germination hole in the composition matrix played a low role in the classification of Impatiens. There were some differences in other quantitative indices among the groups, but there was no difference in the quantitative characteristics of pollen in some Impatiens groups, such as the Impatiens and Racemosae. The combination of Figure 4 showed that the pollen characteristics of Impatiens had some significance in the grouping of Impatiens.

Figure 5.

The situation of the Impatiens, Uniflorae and Fasciculatae groups was relatively clear, but there was a relatively large difference between the two types of pollen in the Scorpioidae group owing to the unique pollen characteristics of I. yui. The similarity of the Clavicarpa was low. Interestingly, there was also a high degree of similarity among the six species of Impatiens that were not grouped. The grouping analysis of the similarity between Impatiens further determined the classification between them, verified the results of systematic clustering and supported the superiority of Impatiens grouping.

Impatiens is rich in germplasm resources, and there are approximately 120 species in the Yunnan–Guizhou Plateau (Luo et al., 2022). In this study of some areas of Yunnan and Guizhou, 48 species of Impatiens were collected, which is, of course, only a fraction of the local species. During the process of collection, the preliminary investigation of Impatiens shows that the there was a high interspecific coefficient of variation of Impatiens, particularly in the variation of flower colour. Three species of Lycos’s with different flower colours, and five species of Lycos’s with different flower colours were found. Their names were marked with 1, 2, 3 and 4 to distinguish them. The variation in height of Impatiens has been confirmed for a long time (Chen, 1978).

We observed the pollen of Impatiens (Figure 1 and Figure 2). The results showed that the pollen of Impatiens was primarily divided into three-groove and four-groove types, and there was a significant correlation between the number of pollen germination pores and its morphology (Figure 4). The pollen characteristics included four pollen germination pores of rectangular and oval shapes and three of triangular circles. Interestingly, Impatiens with three-groove pollen had four sepals, and Impatiens with four-groove pollen had two or four sepals. This phenomenon was used as the difference between primitive and more evolved species of Impatiens plants. As shown, the pollen of the original species of Impatiens was the three-groove type with four sepals; the over-species of Impatiens pollen was the four-groove type with four sepals, and the evolutionary species of Impatiens pollen included the four-groove types with two sepals (Yu, 2012; Yu et al., 2016). The EO of pollen was the reticulate type (Janssens et al., 2012), and the MD of evolutionary species was higher than that of the original species. The addition of reticulation on the pollen surface could contribute to plant pollination and fertilisation and protect the genetic material in plant pollen. The reticulate pattern increases the structural strength and surface friction coefficient of pollen, which helps them interact with insects and increases the ability of insects to carry the pollen (Attique et al., 2022). In our classification based on pollen quantitative characteristics, we found that polar axis length, equatorial axis length and pollen size played an important role in the classification of Impatiens, which was similar to the results of most plant pollen studies (Zafar et al., 2022).

The PCA showed that 11 of the 19 pollen characteristics were more effective than the NS in the classification of Impatiens (Figure 2 and Table 2), indicating that the pollen characteristics of Impatiens play an important role in the classification of Impatiens. Pollen characteristics played an important role in plant classification during the research and development of palynology and have been applied to the classification of many plants (Khansari et al., 2012; Pérez-Gutiérrez et al., 2015; Ullah et al., 2022). During the process of interspecific clustering (Figure 4), the results of pollen classification of Impatiens were satisfactory, but the difference between Impatiens that was highly similar was too low to subdivide some Impatiens that only differed slightly. During the study of pollen morphology, it was also explained that the characteristics of pollen structure can support the classification between genera and higher levels of taxonomy (Umber et al., 2022). We used pollen characteristics as the basis of interspecific classification, and the results showed that, to some extent, pollen characteristics supported the interspecific classification of Impatiens. In summary, the pollen of Impatiens had some positive effect on the classification of Impatiens, whether qualitative or quantitative. However, the identification of specific species of Impatiens also required the support of other characteristics (Lu, 1991). This also indirectly showed that there was a small difference among some species of Impatiens, and the coefficient of variation of Impatiens pollen was not as high as those of other morphological indicators (Lens et al., 2005). This has important reference value for the study of ancient plants and plant evolution (Lens et al., 2012).

Based on the Impatiens grouping system proposed by Yu et al. (2016), the interspecific similarity (Figure 6) and grouping of Impatiens were analysed using pollen morphological characteristics and quantitative data (Figure 7). In this study, the materials that were collected involved two subgenera. There were 33 species of the Impatiens subgenus, which can be divided into six groups. In addition to these groups, six species of Impatiens did not belong to any group. They were designated undetermined groups. In the grouping, the Racemosae group had the most materials with 12 species. According to the interspecific similarity and the grouping of Impatiens, all the plants of the subgenus Clavicarpa were found to be the three-furrow type with a triangular circle. The pollens of Impatiens, Racemosae and Fasciculatae groups were all rectangular or oval. In the Scorpioidae group, there was low similarity between the two species of Impatiens, and they also differed greatly in terms of their shape and size. Among the 35 species of Impatiens, I. yui was the only one with a pollen epidermis. Interestingly, we had not determined that the basic situation of the pollen of the six species of Impatiens was highly consistent. The pollen morphology was pillow-shaped, and it was possible that there was some evolutionary relationship between them. Compared with the other groups, the pollen P/E1 of Impatiens in the Uniflorae group was smaller, and the pollen looked slender. According to the analysis of the grouping form of Impatiens, pollen has been demonstrated to play some role in the grouping system proposed by Yu et al. (2016), which supported the classification of the two subgenera. However, there were some differences in pollen morphology among the seven groups of Impatiens, and the classification system of Impatiens may require further refinement. This is particularly true for the Scorpioidae group.

Figure 6.

Figure 7.

The clustering results of 35 species of Impatiens showed that compared with the results of molecular markers, the clustering results of the original species were partially consistent with those of molecular markers, and the clustering distance of the same class of Impatiens was very short, or directly clustered together. This shows that the pollen morphology is similar to that of molecular markers or other morphological markers in interspecific classification (Yu et al., 2016). With the continuous improvement in the molecular marker technology, increasing numbers of researchers utilised the chloroplast genome (Janssens et al., 2006; Luo et al., 2022) or a gene fragment (Eddie et al., 2003; Swenson et al., 2007; Wang et al., 2014) to study the classification of Impatiens. The species of Impatiens is rich, and it was difficult to make and preserve specimens. The preservation of specimens greatly improved the classification of Impatiens. However, there was only a continuous breakthrough when traditional taxonomy was used, and the classification index was continuously refined. The combination of these taxonomic studies with those that utilised molecular markers and other technology overcame the classification problem of Impatiens and built a more detailed classification system of balsam.

The traditional taxonomy of Impatiens not only includes the observation of pollen by SEM but also involved the classification and analysis of Impatiens using micromorphological features, such as the leaf epidermis (Cai, 2007) and seeds (Martínez-Ortega and Rico, 2001; Janssens et al., 2009; Dadandi and Yildiz, 2015). This was a good way to combine macro- and micromorphologies (Yu et al., 2016) to find a more detailed classification system of Impatiens, but the refinement and integration of classification indicators should be more flawless.