Exploring intraspecific pollen morphology variation in Apocynaceae: A roadmap for horticultural innovation

Apocynaceae is one of the important angiosperm families founded in 1789 as ‘Apocineae’ by Jussieu (El Gazzar et al., 2018). It comprises 375 genera and 5100 species present mainly in subtropical and tropical regions of the world (Endress et al., 2007). Naz et al. (2019) have reported that Pakistan has 19 genera, comprised of 13 cultivated genera (20 species) and 6 endemic genera (6 species). Brown in 1810 separated Asclepiadaceae from Apocynaceae based on pollinium and classified them into three subfamilies (Secamonoideae, Asclepidoideae and Periplocoideae). Schlechter (1905) segregated Periplocaceae based on tetrads pollens from Asclepiadaceae but Endress and Bruyns (2000) classified the family Apocynaceae into five subfamilies, namely those of Rauvolfioideae, Apocynoideae, Periplocoideae, Secamonoideae and Asclepiadoideae. From a global perspective, Asclepiadoideae are comprised of 250 genera and more than 2,500 species; however, in Pakistan, 23 genera and 41 species are presently reported (El-Fiki et al., 2019).

Apocynaceous plants are perennial shrubs, trees or herbs that occasionally grow as annuals, with five joined petals on flowers, astringent milky latex, tufted seeds, and fruits that are typically in pairs (Shah and Ahmad, 2014). The leaves are exstipulate, lanceolate, alternate, simple, opposite decussate, ovate, obovate, oblong, linear, whorled, elliptic or oblong; sessile; or petiolate with an entire or undulate border and an acute apex while occasionally reduced or turned into spines (El-Fiki et al., 2019). Mesophytes and semi-succulent taxa have cylindrical stems, whereas succulent taxa have angled stems. Flowers have traits such as being actinomorphic, hypogynous, hermaphrodite, sympetalous and possessing pentamerous (sometimes tetramerous) whorls with a range of sizes. Further characteristics are a corolla with five joined petals, imbricate lobes and a calyx with five united imbricate sepals; the corona arises from the base of the petals or staminal filaments, and an androecium with five free stamens. The fruit of a parietal placenta consists of two carpels, two free ovaries, two styles that fuse just beneath the capitate stigma, two seeds that generally contain a distal tuft of silky hairs, and two dehiscent follicles containing multiple seeds (Morais et al., 2021).

In Pakistan, it is mostly present in subtropical regions of Mianwali, Khushab (Sakesar), Chakwal, Talagang, Jehlum, Sindh, Rawalpindi, Islamabad etc. in the form of twining shrubs, lianas, perennial herbs, scramblers, vines and also leafless succulent stems present in the order Gentianales. In Apocynaceae, some plants have medicinal values such as diuretic, antidiabetic, anti-inflammatory, stomachic and laxative, as well as utility in the treatment of respiratory disorders (Bahadur et al., 2018). To treat scorpion sting, snake bite and skin disorders, Nerium oleander is mainly used (Ashfaq et al., 2019). Phytochemical analysis reveals that Calotropis procera, Dregea volubilis, Pergularia daemia, Carissa spinarum, Cryptolepis dubia etc. have a significant place, both in traditional and modern medicine (Khan et al., 2022).

Pollen and seed germination tests can be used to identify salt-tolerant crops and pollen grain shapes can be utilised to identify interspecific and intraspecific relationships. These investigations were conducted to ascertain the links, both intraspecific and interspecific, between fruit tree species, which may be significant for processes related to fruit development, fertilisation and pollination (Khaleghi et al., 2019). Breeders and academics have already documented numerous cultivars and genotypes with undesirable pollen. Some cultivars/ genotypes, for example, have sterile pollen or pollen with a low germination percentage. Pollen form and ultrastructure have been linked to growing habitat and pollination biology (Ćalić et al., 2013).

Modern taxonomists are particularly interested in palynological characterisation to distinguish closely related taxa and identification and assessment of interactions among species at several taxonomic levels rely heavily on pollen morphology (Majeed et al., 2023). Palynological studies can help us comprehend plant evolution and phylogeny. Scanning electron microscopy (SEM) is a contemporary technique for studying the micromorphological structure in depth (Majeed et al., 2022). We employ a scanning electron microscope with high-resolution power to examine the surface of pollen grains (Khan et al., 2022; Nabila et al., 2022). SEM is presently known to be one of the most innovative techniques for taxonomic research. Many palynologists are currently using pollen features to precisely and accurately distinguish closely related taxa (Bahadur et al., 2018; Abid et al., 2023).

The study aims to investigate intraspecific variations in pollen morphology within the Apocynaceae plant family. It seeks to provide a comprehensive understanding of the pollen traits that differ among closely related species, offering insights into their genetic diversity and potential applications in horticultural innovation. By creating a roadmap for this exploration, the study intends to contribute to the development of new horticultural techniques and strategies for the Apocynaceae family.

The pollen grains of different plants species belong to different genera, such as Allamanda, Alstonia, Asclepias, Calotropis, Carissa, Cascabela, Catharanthus, Cryptolepi, Dregea, Leptadenia, Nerium, Oxystelma, Pergularia, Periploca, Plumeria, Tabernaemontana and Vincetoxicum. The light microscopic results are shown in Figures 5 and 6, whereas the SEM results in Figures 7–11. The palynological traits of 18 Apocynaceae species that were examined have been summarised in Tables 3 and 4. The major taxonomical characteristics for identifying a species are the pollen size, pollen type, equatorial view, polar view, dispersal unit, aperture ornamentation, exine sculpturing, P/E ratio, exine thickness, polar diameter, equatorial diameter, length of colpi, width of colpi and mesocolpium (Tables 5 and 6).

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Dendrogram and PCA clustering tool analysis

Cluster analysis based on Euclidean distance was performed based on analytical pollen data of selected Apocynaceous species. Two major associations were formed, named Association C1 and Association C2. Association C1 consists of three species. These species have similarities based on colpi length and width and exine thickness. Association C2 consists of 15 species and these associations have similarities based on mesocolpium distance, polar and equatorial distance, and the P/E ratio, as shown in Figure 11. PCA analysis was used on the pollen quantitative mean data of Apocynaceae species to determine the variance among different variables in the context of eigenvariables. PCA illustration showed that the length of colpi on the PC1 axis of A. curassavica and D. volubilis was characterised by a higher exine thickness, whereas on PC2 a higher value in terms of mesocolpium distance was observed with reference to V. spirale (Table 7 and Figure 12).

Figure 12.

Table 7.

PCA % variance loadings for the Apocynaceous pollen.

PC	Eigenvalue	% variance
1	2.35994	39.332
2	1.53998	25.666
3	1.0319	17.198
4	0.82796	13.799
5	0.239496	3.9916
6	0.000728	0.012128

PCA, principal component analysis.

The study on intraspecific pollen morphology variation in Apocynaceae is of significant importance as it provides a roadmap for horticultural innovation. Understanding these variations can aid in breeding programmes, helping to develop more resilient and productive cultivars. This research can also contribute to conservation efforts, as it aids in the preservation of genetic diversity within the family. Ultimately, the study’s findings have far-reaching implications for both horticulture and biodiversity conservation.

In the present study, the palynological traits of 18 Apocynaceae species were studied quantitatively and subjectively with the use of microscopy. The systematic study of palynomorphological features in flowering plants at higher taxonomic levels has considerably benefitted from the use of both a light and a scanning electron microscope (Al-Hakimi et al., 2015). Based on the traits of pollen that were studied, a taxonomy key was developed for fast and precise identification. Apocynaceae identification at the species level is too difficult because of its increased diversity and variable pollen and pollinia forms. The primary objective of this work was to morphologically evaluate the Apocynaceae family using pollen and pollinia to identify traits that would be helpful for taxonomic classification.

Previous studies that attempted to provide important and useful palynomorphological data on this family were unsuccessful in combining a comprehensive approach to pollens and pollinia in a single investigation. In this study, we present palynological studies on the Apocynaceae, which would help taxonomists correctly identify several highly significant plants. When examining pollen, microscopy is crucial for comprehending the taxonomic significance of plant species within families (Raza et al., 2020).

Colpi, mesocolpium, translator, corpusculum, exine sculpturing, aperture ornamentation, exine thickness, pollen type, size, polar and equatorial views, pollen sac length and width, pollinium ornamentation, pollinium shape and many other features were examined using SEM. These palynological characteristics are essential for correctly classifying different species in plant taxonomy (Ur Rahman et al., 2019).

The pollen grains and pollinia of the family Apocynaceae exhibited a considerable degree of morphological variety. Pollens ranged in size from small to quite large, and tricolpate to tetracolporate. Pollen shapes ranged from sub-spheroidal, to oblate spheroidal to rhomboidal, while exine sculptures ranged from psilate, through scabrate, to rugulate. Polar views were lobate, isopolar to irregular. On the other hand, in pollinia, the orientation was pendent to transverse, the shape was obovate to narrowly oblong, sterile margin was mostly absent and translator attachment to corpusculum was sub-apical to apical. The pollen of A. cathartica was described as sub-oblate, prolate, isopolar, having radial symmetry and having a granulate tectum in earlier research. In contrast, A. scholaris pollens had sub-circular to obtusely triangular apertures in polar view and were medium-sized, oblate spheroidal, colporate and characterised by scabrate exine sculpturing (Meena et al., 2011). This is not the case for the pollen grains of A. scholaris, which were found to be small, tricolporate, sub-spheroidal, lobate and isopolar, and to have psilate to perforate exine sculpturing. In contrast, the pollen grains of A. cathartica were found to be medium in size, tricolporate, elliptic and triangular (convex), and to have reticulate exine sculpturing.

In the present research, C. macrocarpa and C. spinarum pollen grains were medium, tricolporate, oblate sub-spheroidal, circular, lobate and isopolar, and shed as a monad. Exine sculpturing was perforated, and psilate to regulate has been observed. In C. thevetia, pollen grains were very large, tricolporate, prolate or sub-spheroidal, and lobate, and shed as a monad. Exine sculpturing with the characteristics of microreticulate to perforate has been observed (Naz et al., 2019). C. roseus and C. dubia pollen grains were large, tricolporate, oblate spheroidal and irregular; exine sculpturing was perforate to scabrate and tetrads; and rhomboidal, reticulate exine sculpturing has been detected, which represents a variation from the findings in Zafar et al.’s study (2022). Pollen grains in N. oleander and T. divaricata were medium, tricolporate, oblate spheroidal, and circular, while exine sculpturing was psilate and perforate, respectively. Pollen grains in P. rubra were medium in size, tricolpate, prolate sub-spheroidal, round and isopolar, and exine sculpturing was observed to be a perforate in regulation; these findings demonstrate a good consistency with those of Mallick’s study (2020). P. aphylla pollen ranged in size from small to large, the shape of the pollen was tetrad, the polar view was rhomboidal while the equatorial view was oblong, pollen shed as tetrad and psilate exine sculpturing was detected; however, these interpretations vary (Swarupanandan et al., 1995).

In A. curassavica, pollinia were large, brown, pendent and narrowly oblong, sterile margins were absent, translator attachment to pollinium was basal, corpuscular arm was absent, translator attachment to corpusculum was sub-apical and psilate perforate exine sculpturing was present. In C. procera, pollinia were large, canary yellow, pendent and obovate, sterile margins were absent, translator attachment to pollinium was basal, corpuscular arm was absent while translator attachment to corpusculum was sub-apical, and the pollen type was polyads. On the other hand, in L. pyrotechnica, pollinia were medium, brown, transverse and orbicular, sterile margins were present, the orientation of sterile margins was apical, translator attachment to pollinium was basal and corpuscular arm was absent while translator attachment to corpusculum was apical; these outcomes confirm the interpretations derived in the study of Shah and Ahmad (2014). In O. esculentum and P. daemia, pollinia were large, yellow, pendent and narrowly oblong, sterile margins were absent, translator attachment to pollinium was basal, the corpuscular arm was absent, translator attachment to corpusculum was apical and pinkish brown, obovate, pseudo-sterile margins were present and the corpuscular arm was absent while translator attachment to corpusculum was apical, respectively. In D. volubilis, pollinia were large, sulphur yellow, erect and reniform, sterile margins were absent, translator attachment to pollinium was basal and corpusculum arm was absent while translator attachment to corpusculum was apical. These results were highly distinguishable from those of Pal and Mondal (2019). In V. spirale, pollinia were canary yellow, pendulous and corpusculum ovate, and oval pollinial sac confirms the findings of Yaseen and Perveen (2014).

The morphopalynological study linked to horticultural exploration harnesses the knowledge that contributed to the development of more resilient and productive horticultural practices within the Apocynaceae family.

Comparative studies of pollen structures among Apocynaceous taxa reveal that the taxonomy of exine wall characteristics has raised scholarly interest in pollen biology. This research focusses on the qualitative and quantitative micromorphological microscopic features of 18 Apocynaceae pollen. The microscopic authentication of closely related pollen characters of species is identified based on pollen taxonomy. The maximum mesocolpium distance was noted in C. roseus (44.25 μm) and the minimum in T. divaricata (14.1 μm). Exine sculptured tectum was examined as reticulate regulated and perforated. This work shows that palynology is very dependable in reconstructing historical vegetation and decrypting taxonomic links based on microscopic visualisation of pollen micromorphology and serves for accurate species identification. This study’s findings on intraspecific pollen morphology variation in Apocynaceae provide valuable insights for horticultural innovation, enabling targeted breeding and cultivation strategies for improved plant species.