The flowering plant genus; Hypericum L. is one of the 100 large angiosperm genera and the largest genus of nine genera of the family Hypericaceae with about 469 species occurring naturally and have been introduced to, every continent except Antarctica. Approximately 80% of the diversity of the family is within Hypericum    . Family Hypericaceae belongs to order Malpighiales and contains well-known medicinally valuable species   . The genus Hypericum is known worldwide for its traditional and modern uses. There are variety of bioactive compounds found in many species of Hypericum, which are used pharmacologically as well as to treat many diseases like tumors, cancer, AIDS, mild depression and kidney failure etc.    . The most well-known species of the genus Hypericum is St. John’s wort Hypericum perforatum which is economically important crop plant with antiviral, anticancer, antibacterial and antidepressant properties   . Flowers of this plant are known to use for relieving body pains and tea preparation in Kaghan Valley  .
Genus Hypericum L has been studied by various researchers for its taxonomic, chemotaxonomic, pharmacological, karyological, ethnobotanical aspects. However a recent phylogenetic study based on morphological characters was carried out (Nurk and Blattner,  while based on phylogenetic characters was studied by (Nurk  and Rhufel et al.  . Palynology of the Hypericum recently studied by Otaghvari et al. and previously have been studied by Khan, Thomas, Aytug et al., Charpin studied regarding allergic pollen), Yi, Clarke, Barros and Ramos, Martonfi et al., Meseguer and Sanmartin and Ocak et al.,   -  but the pollen morphology of Hypericum from Pakistan is still not known. However it has been largely studied for its medicinal properties, ethnobotanical uses, traditional knowledge and antioxidant capacity    .
The knowledge of pollen morphology and taxonomy can be used as an instrument of multiple scientific researches in systematic botany, paleobotany, paleoecology, pollen analysis, aeropalynology, criminology, allergy, stratigraphic correlation of oil-bearing rocks and coal fields, drugs in the field of medicopalynology and improvement of honey in the field of mellittopalynology and copropalynology  . Application of pollen morphology in plant taxonomy is best evidence in the flowering plants, especially in the angiosperms. The largest variety of pollen morphology occurs among the angiospermic plants  . Like other disciplines pollen grains have an important part in modern issue of plant taxonomy  .
The present investigation is based on pollen morphology and pollen fertility estimation of three medicinally important plant species of genus Hypericum studied for the first time from Pakistan.
2. Materials & Methods
Study Area―The Kaghan valley covering an area of 19790.4 hectare spreads on either bank of river Kunhar, situated in the district Mansehra, Hazara division, Northern Pakistan (Figure 1). It is located between 34˚ - 14' and 35˚ - 11' north latitudes and 72˚ - 49' and 74˚ - 08' east longitude. It is bounded in the north by Batagram and Kohistan districts, in the east by Muzafarabad district of Azad
Figure 1. Map of the study area.
Jamu and Kashmir, in the south by Abbottabad and Haripur districts and in the west by Swat district  . The climate of the the Kaghan valley is cold in summer owing to snow clad mountains and very cold in winter Temperature ranges from 2˚C - 30˚C in the district The general vegetation is of the scrub type which includes the shrubs or medium size trees  . There is a great variety of medicinal plants growing throughout the area which are commonly being used by the local people.
Three medicinally important plant species of genus Hypericum L. were observed for their pollen morphology and pollen fertility estimation in the Plant Taxonomy Laboratory, Herbarium of Quaid-i-Azam University, Islamabad & Centralized Science Laboratory University of Karachi. Pollen materials of fresh specimens were collected from flora of Kaghan Valley and used for LM and SEM.
3. Light Microscopy (LM)
For light microscopy slides were prepared by using glycerin jelly and safranine according to modified method of Mazari et al.  . The slides were prepared and observed at different magnification. The micro-graphs of these mounted materials were taken using a digital camera (Meiji CCD Model 00179048, Canada) fitted on the Lieca light microscope (DM 1000, Germany). All photographs were taken on different object lenses by using oil emulsion.
4. Scanning Electron Microscopy (SEM)
The pollen grains were prepared for scanning electron microscopy (SEM). Pollen grains suspended in a drop of water were directly transferred with a fine pipette to a metallic stub using double sided cello tape and coated with gold in a sputtering chamber (Ion-sputter JFC-1100). Coating was restricted to150A. The S.E.M examination was carried out on a Jeol microscope JSM-2.
5. Pollen Fertility Estimation
Pollen fertility percentage was calculated on the basis of staining by Glycerin jelly. The prepared slides were observed at low magnification. Glycerin jelly was used in pollen staining and prepared according to modified method of Mazari et al.  . Percentage of pollen fertility was calculated on the basis of staining by Glycerin jelly. The number of stained and unstained pollen grains was counted. Fully stained pollens were considered fertile while the lightly stained or unstained and deformed pollens were considered sterile. Percentage of pollen was determined by the formula: No. of total pollen/ No. of fertile pollen = Percentage Pollen fertility.
The descriptive terms for pollen morphology are used according to terminology of Erdtman), Kremp, Faegri & Iversen, Punt et al., and Walker & Doyle  -  .
The pollen morphology and pollen fertility estimation of the three selected species of Hypericum was carried out by using light and scanning electron microscopy. Micrographs of pollen grains are presented in Figure 2 and Figure 3. Findings of each species are represented in an order of local name, English name, phytogeography, flower color and season and palynomorph features. These results are summarized in Table 1.
(a) (b) (c) (d)
Figure 2. Light micrographs of pollen grains of Hypericum L. (a), (b): Hypericum elodeoides: Polar view, (a) (b) equatorial and polar view; (c), (d): Hypericum oblongifolium: Polar view, (c) equatorial view, (d), (e), (f): Hypericum perforatum: Polar view, (e) equatorial view, (f) (1000×).
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 3. Scanning electron micrographs of pollen gains of Hypericum L. (a)-(c): Hypericum elodeoides: Polar view, (a) equatorial view, (b) exine sculpturing, (c) (d)-(f): Hypericum oblongifolium: Polar view in bunches, (d) equatorial view, (e) sculpturing, (f) (g)-(i): Hypericum perforatum: Polar view, (g). Equatorial view (h). Sculpturing, (i).
6.1. Hypericum elodeoides Choisy
Local Name: Kasni.
English Name: Marsh Stewart John’s wort.
Phytogeography: It is found from Kashmir to Yunnan and Burma.
In Pakistan it is found in Basaoli, Budrawur, Gulmarg, Kashmir & Northern areas.
Flower color & period: Yellow & July-September.
Palynology: The pollen is monad, diad and tri to tetra-colporate. The shape of pollen in polar view is circular to semi-angular and in equatorial view is oblate-spheroidal (Figure 2(a), Figure 2(b)). The polar axis (P) is 19.91 µm (17.5 - 22.5 µm) and equatorial diameter is 20.6 µm (19 - 22.5 µm). P/E ratio is 0.96 µm. Exine thickness is 1.41 µm (1.25 - 1.5 µm) (Table 1).
Sculpturing: Foveolate with depressions less than 1 µm in diameter (Figure 3(c)).
6.2. Hypericum oblongifolium Choisy
Syn: H. cernuumRoxb., nomen
Local Name: Pinli
English Name: St. John’s wort.
Phytogeography: It is found in Western Himalayas from Kurram to Nepal.
In Pakistan it is found in Kurram, lower Swat, Hazara, Malakandi, Kaghan, Nathiagali, Muree, Kashmir, Poonch.
Flower color & period: Yellow & March-August.
Palynology: The pollen is monad and tri-colporate. The shape of pollen in polar view is circular to semi-angular and in equatorial view is oblate-spheroidal (Figure 2(c), Figure 2(d)). The polar axis (P) is 23.0 µm (20 - 27.5 µm) and equatorial diameter is 25.35 µm (23.75 - 27.5 µm). P/E ratio is 0.90 µm. Exine thickness is 1.58 µm (1 - 2 µm) (Table 1).
6.3. Hypericum perforatum L.
Local Name: Bilsana.
English Name: Goat weed /Tipton’s weed/ Klamathweed /St. John’s wort.
Phytogeography: Europe and N. Africa to W. China and N.W., India. Introduced to East Asia, America. Australasia and South Africa.
In Pakistan it is found in Kurram, Chitral, Swat, Astor, Gilgit, Balistan, Hazara, Muree Hills, Poonch & Kashmir.
Flower color & period: Yellow & June to September.
Table 1. Summary of qualitative and quantitative characters of pollen of Hypericum taxa.
Note: All the measurements are in µm. (P = Polar Diameter, E = Equatorial Diameter, P/E Polar diameter divided by Equatorial diameter.
Palynology: The pollen is monad and tricolporate. The shape of pollen in polar view is circular to semi-angular and in equatorial view is oblate, prolate and rhomboidal (Figure 2(e), Figure 2(f)). The polar axis (P) is 20.78 µm (17.5 - 21 µm) and equatorial diameter is 12.53 µm (17.5 - 38 µm). P/E ratio is 1.65 µm. Length of colpi is 4 µm (5.75 - 8.25 µm) and width of colpi is 2.37 µm (2.25 - 2.5 µm). Exine thickness is 1.35 µm (1 - 2 µm) (Table 1).
Pollen fertility: Pollen fertility is valuable for the taxonomists in attempting to distinguish putative hybrids from the parent plants and is also useful to determine the degree of fertility in those plants that are grown under unfavorable conditions  . The pollen fertility studies have been proved helpful for recognizing wide range of variation existing within plant species and differentiating plant species within genera. Many workers have tried to correlate in vitro pollen fertility with a survival of a plant under its natural habitat  . Matin et al.  studied pollen fertility of 50 medicinal plants of Shogran Valley. Fertility ratio of the plants ranged from 98% - 100%, which indicated that flora of Shogran was well established and stable. In the present study among the 3 species of Hypericum; pollen fertility percentage ranged from 82.66% to 90.90%. It indicates that Hypericum species from Kaghan valley is well established and the species are stable in the area.
In recent studies on palynology of Hyperium by Otaghvari et al., Martonfi et al. and Ocak, et al.    the regular and irregular pollen grains have been discussed. In the present study pollen examination of selected Hypericum taxa was conducted from Pakistan for the first time. Interesting findings were found regarding pollen characters of selected species. The pollen fertility estimation of the species was also investigated. Generally pollen grains were small, monad and isopolar. The type of pollen grains 3 - 4 colporate in H. elodeoides while 3-colporate in other two species. In polar view in all species pollen shape was circular to semi-angular (Figure 2(a), Figure 2(c) and Figure 2(e)). But in equatorial view the pollen grains were oblate-spherioidal in H. elodeoides and H. oblongifolium but prolate H. perforatum (Figure 2(b), Figure 2(d) and Figure 2(f)). The present study showed palynological variation among these three species of Hypericum especially in exine sculpturing of pollen grain which was foveolate sculpturing in Hypericum elodeoides, reticulate in H. oblongifolium and scabrate in H. perforatum. Some of our findings regarding shapes and small to medium size of pollen grains are in accordance with  pollen grains but in contrast with regard of exine sculpturing which is different in all species studied. Similarly palynologiacal analysis of seven Hypericum taxa were studied by  , by light and scanning electron microscopy. According to them there were no essential differences in the exine structure of seven Hypericum species. The present showed much variation in regard of pollen shape in equatorial view and sculpturing pattern of exine which was different in all species studied. Pollen grains are mainly classified on the basis of their shape, size, aperture, polarity, symmetry and exine sculpturing. Along with these characters; exine sculpturing pattern is of great importance from phylogenetic and evolutionary point of view  . The present study showed that the studied palynological characters would be more useful for further palynological, karyological and taxonomic studies of Hyperium species.
We are thankful to PCSIR Karachi for financial support for SEM and to Centralized Science Laboratory (CSL), University of Karachi for assistance in Scanning electron microscopy. We are also grateful to Department of Plant Sciences, Faculty of Biological Sciences Quaid-i-Azam University Islamabad.
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