Ecological acclimation on the pheno-characters of Ginkgo biloba L. outside its native range: Perspective to pollen grains, Egypt

J. Bio. & Env. Sci. 20 23 65 | Taia et al. RE RERE RESEARCH SEARCHSEARCH SEARCH PAPER PAPERPAPER PAPER OPEN ACCESS OPEN ACCESSOPEN ACCESS OPEN ACCESS Ecological acclimation on the pheno-characters of Ginkgo biloba L. outside its native range: Perspective to pollen grains, Egypt Wafaa K . Taia, Selim Z . Heneidy, Laila M. Bidak, Amal M . Fakhry, Soliman M . Toto * Department of Botany & Microbiology, Faculty of Science, Alexandria University, Alexandria, Egypt Article published on August 07, 2023 Key words: Acclimation , Ginkgo biloba , Gymnosperm , Male cone, Phenology, Pollen grains Abstract A fertile branch at three meters high was chosen from a 25-year-old Ginkgo biloba male tree for this investigation. The tree was examined daily to record the suitable time of mature yellow catkins (male cone), which was in April 2023. Pollen grains from the mid catkin from the nine fertile nodes beside those collected from the four Earths directions have been examined carefully by both the light and Scanning electron microscopes. Element contents have been investigated within the different pollen stages. The data obtained revealed that most of the investigated pollen pheno-characters showed highly significant differences between the catkin developmental stages and in between the different positions in each catkin stage, except within few characters. Despite the state of differences recorded within the different positions of the catkins there were highly significant differences between the different nine stages of the catkin position. This investigation showed that G. biloba trees growing in Alexandria city are adapted to the city climate and release their pollen grains gradually to ensure successful pollination. The microsporangia mature gradually; accordingly, their pollen grains are in different developmental and hydrolytic states. The obtained results proved the high adaptation processes which are shown in both the phenolmorphological states beside their mineral contents of the G. biloba pollen grains. * Corresponding Author: Soliman M. Toto  [email protected] Journal of Biodiversity and Environmental Sciences (JBES) ISSN: 2220-6663 (Print) 2222-3045 (Online) Vol. 23, No. 2, p. 65-77, 2023 http://www.innspub.net J. Bio. & Env. Sci. 20 23 66 | Taia et al. Introduction Ginkgo biloba is a mysterious tree; it is the oldest living gymnosperm tree which overcomes much generic extinction, and it is the only species still adapt with all the environmental disorders from the Ginkgophyta (Zhou & Zheng, 2003). It is considered as a unique tree in many aspects, it occupies its unique taxonomic division, class, order, family and genus with great genetic distances with its relatives beside its important food content, and medicinal values. This species is considered a living fossil as it overcomes many global catastrophes and adapts with great environmental and climate changes. This species has many forms of adaptation which made it modulate with the environmental disturbance meanwhile it has its specific way of fertilization. It produces huge amounts of light pollen grains to succeed its anemophilous mode of pollination. G. biloba pollens carried inside microsporangia in subsequent degrees of development to insure long fertile time (Lu et al., 2016). After pollen dispersal, the pollen grain releases water and becomes typically folded inwards in its aperture or leptoma to keep the exposed aperture area in hydrated state (Hesse et al., 2009). Worth noticing that the shape of dry pollen grains after dispersal is bilaterally symmetrical with monosulcate wide aperture and nearly smooth exine ornamentation (Lu et al. 2011a). After hydration with the pollination drop, the pollen converts into a round shape (Tekleva et al., 2007). This pollen shape change is considered as a mode of adaptation to keep more pollen grains in fertile and hydrated form. Trees of G. biloba are native to China and introduced to Japan, Europe, and North America (Del Tredici 1991 & 2000; Tsumura et al., 1992). Its trees are commonly planted in the parks as ornamentals for their woody trunk and characteristic leaves. The trees are dioecious i.e., the female megasporangia carried in trees separate from those who carried the microsporangia. The microsporangia are carried in an inflorescence called catkin. The catkins originated from the axils of the leaves on short shoots and their numbers differ from node to another. Each sporangiophore consists of a stalk and sterile extension which bears two pendant pollen sacs and release their pollens by the aid of longitudinal slit (Klimko et al., 2016). Pollen morphological characteristics have been studied in detail as they are essential for successful fertilization and reproduction in G. biloba. This study presents information on aspects of pollen development of G. biloba, which are relevant to understanding its ecological characteristics as an introduced species in Egypt. Meanwhile to find out how much the catkin-like developmental stages affect the pollen morphological characters and its hydration state which in turn affect its fertility to help in the process of the species conservation and reproduction. In addition, to highlighting the special strategy of G. biloba to acclimate with environmental factors outside its native range with respect to pollen grain characters. Materials and methods Study Species Male reproductive structures were studied for about 25-yr-old male trees of G. biloba, grown in the Botanic Garden of Alexandria University in Alexandria city, Egypt. Catkin-like inflorescences (male cone) were collected by the authors. Trees were investigated daily to record the suitable time with yellow male cones indicating mature microsporangia, which were in April (2023). A fertile branch at elevation of about three meters high was chosen for this study. The mid catkin from the nine fertile nodes beside those collected from the four directions (North, East, South and West) have been dealt with in this study. Three positions from each catkin have been chosen; near the peduncle (L), mid the catkin (M) and near the tip (U) (Fig. 1 A & B); and carefully smeared onto glass slide with drop of glycerol jelly for light microscope examination. Counting the total number of pollen grains, number of hydrated (Spherical) and dehydrated (Prolate or Oval) in each position from each node, pollen measurements and photographed using OPTICA (B150D) light microscope fitted with USB digital-Video Camera and Computer Software at 10X40 lenses. The polar, equatorial axis or radius as well as the exine J. Bio. & Env. Sci. 20 23 67 | Taia et al. thickness and aperture measurements were recorded in at least 30 pollen grains to obtain the minimum, maximum and calculate the mean and standard deviation of each item. Fig. 1. A-The nine studied stages BThe three Position in each catkin (L, M. U). [Photos taken by Heneidy S. and Toto S.] SEM investigation and mineral contents Non-acetolysed pollen grains have been sputtered onto cleaned, Aluminum labeled stubs, coated with 20 nm Gold in a Polaron JFC-1100 coating unit, examined and photographed using JEOL-JSM.I T200 Series Scanning Electron Microscope allocated in the electron microscope unit, Faculty of Science, Alexandria University, Egypt. Pollen mineral contents were measured in pollen grain pellets and subjected to X-ray analysis under 20 kv using the same SEM. The pollen terminology was adopted from Faegri and Iversen (1989), while the shape of the pollen grains followed that of Erdtman (1952). Study area The studied trees of G. biloba are grown the Botanic Garden of Alexandria University in Alexandria city in Northern Egypt. Alexandria is the second largest city in Egypt. It is located in the northern Mediterranean coastal region of Egypt. In general, Egypt has arid climate, but Alexandria has its characteristic climate. It has hot, humid weather during the summer, with the hottest months are July and August and cool rainy weather during the winter (Fig. 2) Fig. 2. The mean temperatures from 1991 till 2020 in Alexandria, Egypt. Show the average temperatures in Alexandria in Celsius. Data analyses Quantitative data were expressed as mean and standard deviation for normally distributed quantitative variables. One way ANOVA test was used for comparing the different studied groups and followed by Post Hoc test (Tukey) for pairwise comparison. The significance of the obtained results was judged at the 5% level. Five replicas for each group and the data were expressed using Mean ± SD. Results The chosen branch had nine fertile nodes with one or two sterile nodes in between. Each fertile node has from three to five catkin-like inflorescences (Fig.1 A). The micro-sporophylls are in spiral arrangement and bear two fertile micro-sporangia. Each microsporangium contains different total numbers of pollen grains and different ratios between the hydrated (Spherical) to the dehydrated (Oval or Prolate) (Table 1). J. Bio. & Env. Sci. 20 23 68 | Taia et al. These variations recorded in between the position of the micro-sporangia of the same catkin. The ratio between the spherical pollen grains to the oval ones (S/P) was greater in the low micro-sporangia; near the peduncle; than the middle and upper ones. Catkins from the ninth node and those from the four main directions; mid and upper positions had S/P pollens greater than the lower position (Fig. 3, 4, 5). Fig. 3. Total number of pollen grains Anine studied stages, B-four directions Fig. 4. Ratio between spherical and prolate Astudied stages, B-four directions. Fig. 5. Shape of pollen grains according to P/E ratio Anine studied stages B-four directions. Pollen grains morphology Light microscope investigation The pollen grains by the first investigation; under the light microscope (Fig.6-17 and Table 1); appear either apolar or isopolar, bilateral symmetric, spherical (P/E=1), oval (P/E˃1) or subprolate (P/E±1.2). The radius of the spherical pollen grains varied between 21.3µm in the lowest micro-sporangia on the 1 st . node near the peduncle, while it reaches over 26 µm in the upper nodes (8 & 9) as well as in the East (M) and North (Up) directions (Fig. 15). Apertures are either mono-aperturate (Fig. 6, 8) or anaporate (Fig. 12, 15, 16) in the spherical pollens. They are mono-sulcate in the oval or subprolate pollen grains (Fig. 7, 9, 10, 11, 14). Under the light microscope, the apertures appear as bi-colpate and this is due to the wide opening (Fig. 13, 17). The exine is considerably thin it ranges from 0.5 to 0.8 µm thick which appears as punctuate or ornamented with small granules under the light microscope (Fig.7,9,11,14,15,16). J. Bio. & Env. Sci. 20 23 69 | Taia et al. Fig. 6-17, LM photographs. Bar = 20 µm. Fig. 6 stage 1L spherical and subprolate pollen grains, Fig. 7 stage 1M spherical and subprolate pollen grains with wide sulcus, Fig.8 stage 2L spherical pollen grains with anaporate aperture, Fig.9 stage 5M prolate pollen grains with wide sulcus, Fig.10 stage 5U spherical and prolate pollen grains with narrow sulcus, Fig.11 stage 7L spherical and prolate pollen grains with narrow sulcus, Fig.12 stage 6U spherical pollen grain with anaperturate aperture, Fig.13 stage 8M prolate pollen grains with two slit like apertiures, Fig.14 stage 9U subprolate pollen grains with wide and slit-like apertures, Fig. 15 stage EM spherical and subprolate pollen grains with wide sulcus, Fig.16 stage NL spherical pollen grains with anaporate aperture, Fig.17 stage WL prolate pollen grains with two slitlike apertures. Scanning Electron Microscope investigation (SEM) Under the SEM investigation the detailed features of both the apertures and exine ornamentation have been clarified (Fig. 18-30 and Table 1). The apertures are mono-sulcate in the oval pollen grains with unidentified margo (Fig. 18, 25). The sulcus is almost ellipsoidal and extends till near the poles (Fig. 18, 23, 25). The spherical pollen grains have very wide nearly rounded aperture called anaporate (Fig. 28, 29). The sulcus always extends from distal to proximal poles and its apex is either round (Fig. 18) or sharp (Fig. 23, 25). The sulcus membrane is psilate (Fig. 19, 22). The exine ornamentation differs between the studied pollen grains, from the psilate or faintly rugate (Fig. 20, 21) to ulcerate (Fig. 27, 30) or even tectate perforate ornamented with small granules (Fig. 26). In a few pollen grains, whatever the developmental stage or directions, the tectum ornamented with rod-shaped extensions (Fig. 23, 24). Fig.31. Elements in the different pollen shapes and positions in the nine studied stages Pollen grains mineral contents The mineral contents in representative developmental stages show that all the pollen stages shared the elements C, O, mg & K in abundant contents. J. Bio. & Env. Sci. 20 23 70 | Taia et al. The two elements; P & S; present in trace amounts in all the stages except the 1 st , 8 th and 9 th stages (P 0.77 in stage 6 to 1.2 in stage 2 and S 0.15 in stage 4 t0 0.3 in stage 2). Traces of Na (0.19) recorded in the first stage only and Cu & Zn recorded in the 1 st , 8 th and 9 th stages only with small amounts (2.93 & 1.65; 4.12 & 2.31; 5.53 & 2.97 respectively). Traces of Ca recorded in the 2 nd , 6 th and 7 th stages only (0.42, 0.29, 0.21 respectively), while Al recorded in trace amount (0.95) in stage 9 (Table 2 and Fig.31). Table 1. Number of mature catkins, total pollen grains count in five microscopic fields, ratio between spherical/prolate pollens, radius, or polar and equatorial axis lengths in µm: min-max (mean ±SD) and ratio between polar and equatorial axis. St No of Ca TPG SphPG PrPG Exine Radius Pore width PAL EAL Sulcus L Sulcus W Thickness Orn 1 L 3 7-10 7.2±2.95 21.3-24.6 23.5±1.98 18.3-18.6 18.5±0.08 0.0-24.5 19.4±5.82 0.0 - 23.5 18.4±4.8 2 22.2-24.5 21.4±3.62 2.2 - 4.1 3.2±1.0 8 0.5-0.6 0.58±0.05 P or FR 1 M 15-48 29.6±16.01 24.0 - 25.2 24.7±0.0 8 15.2-15.8 15.5±0.18 0.0 - 26.8 14.97±13.6 8 0.0 - 25.0 14.4±13.1 7 22.8-26.8 24.7±2.68 1.4 - 2.9 2.2±0.6 3 0.5-0.6 0.58±0.05 1 U 16-52 43.6±9.5 24.7 - 25.2 24.94±0.1 8 20.7 - 21.2 20.84±0.3 8 0.0 0.0 0.0 0.0 0.06 2 L 5 5-24 19.8±6.11 24.8-25.1 24.9±0.32 20.8-22.1 21.9±0.22 0.0 - 24.5 14.0±10.6 6 0.0 - 23.6 14.0±9.5 5 22.3-24.5 22.9±1.96 1.8 - 3.9 2.5±1.3 8 0.5-0.6 0.58±0.05 R 2 M 5 - 26 15.4±10.21 24.2 - 24.5 24.4±0.13 19.2 - 21.5 20.6±1.13 0.0 0.0 0.0 0.0 0.6 - 0.8 0.68±0.11 2 U 9-13 11.2±1.48 22.9-24.5 23.9±0.75 19.9-22.5 20.5±1.75 23.1-26.6 24.8±1.22 19.6-24.4 21.7±1.90 23.1-26.6 24.8±1.22 1.6 - 4.2 3.5±0.6 7 05-06 0.58±0.05 3 L 4 6-25 15.8±8.08 24.5-25.3 24.9±0.31 20.2-22.9 21.6±1.31 0.0-26.4 25.8±1.02 0.0 - 22.2 12.8±11.6 7 24.6-26.4 25.8±3.28 2.0 - 4.8 3.7±1.0 8 0.6 FR 3 M 18 - 58 (39.8±17.7 0) 24.0-25.3 24.7±0.61 20.0-22.3 21.0±1.31 24.6-26.5 25.6±0.72 21.5-24.4 22.7±1.28 24.6-26.5 25.6±0.72 2.8 - 4.8 3.9±0.7 3 0.6-0.8 0.68±0.11 3 U 19-42 33.4±8.74 24.0-24.7 24.4±0.26 19.8-21.7 20.2±1.36 25.2-26.0 25.6±0.33 23.8 - 25.2 24.2±0.6 2 25.2-26.0 25.6±0.33 2.0 - 3.8 3.2±0.6 2 0.6 4 L 4 14-33 24.8±7.40 25.4 - 27.6 26.1 ±0.96 22.2-23.6 23.1 ±0.42 26.0-28.0 26.9±0.84 22.9 - 23.7 23.2±0.5 0 26.0-28.0 26.9±0.84 1.8 - 2.6 2.0±0.5 8 0.5-0.6 0.56±0.06 FR or TP wRSsT 4 M 14-26 19.4±6.31 24.8-25.6 25.2±0.36 20.2-21.9 21.2±0.66 23.2-26.6 24.9±1.33 21.0-22.9 22.1±0.97 23.2-26.6 24.9±1.30 2.0 - 3.2 2.4±0.7 7 0.6 4 U 18-28 23.8±4.03 24.6-25.3 24.9±0.27 20.6-21.3 20.7±0.77 24.4-27.5 25.6±1.2) 22.4 - 23.2 22.8±0.3 3 24.4-27.0 25.5±1.22 2.0 - 4.6 3.2±1.3 8 0.6-0.8 0.68±0.11 5 L 4 4-14 9.8±4.15 24.4 - 25.4 24.9±0.4 8 20.2 - 21.8 (20.2±1.4 8) 24.9 - 25.8 (25.2±0.46 ) 21.1 - 22.4 (21.9±0.3 8 24.9 - 25.8 (25.2±0.4 6) 2.4 - 4.8 3.6±1.1 7 0.6-0.8 0.68±0.11 P or TP wRSsT 5 M 3 - 9 6.8±2.39 0.0 0.0 25.4 - 26.2 25.8±0.30 21.9 - 23.4 23.0±0.4 25.4 - 26.2 25.2±0.30 2.2 - 3.2 2.5±0.7 0.6 - 0.8 0.68±0.11 J. Bio. & Env. Sci. 20 23 71 | Taia et al. St No of Ca TPG SphPG PrPG Exine Radius Pore width PAL EAL Sulcus L Sulcus W Thickness Orn 7 2 5 U 10-18 14.0±3.39 24.2 - 24.8 24.4±0.3 8 21.8-22.6 22.0±0.88 23.7-25.7 24.7±0.86 20.8-21.5 21.1±0.33 23.7-25.7 24.8±0.86 3.6 - 5.2 4.4±0.7 7 0.6-0.8 0.68±0.11 6 L 3 5 - 19 13.8±5.07 24.2 - 25.3 24.7±0.43 20.2 - 22.3 21.2±1.03 0.0 0.0 0.0 0.0 0.6 Ulcerat e 6 M 3 - 6 4.6±1.34 23.9 - 24.9 24.6±0.39 20.6 - 22.5 21.2±1.39 0.0 0.0 0.0 0.0 0.5 - 0.6 0.54±0.05 6 U 3 - 9 6.6±2.35 23.5 - 24.5 24.1±0.36 20.2 - 22.6 21.2±1.36 0.0 0.0 0.0 0.0 0.5 - 0.6 0.54±0.05 7 L 4 3-8 5.6±1.95 24.9-25.3 25.1±0.15 20.4-22.3 21.1±1.22 0.0-26.8 12.4±14.06 0.0-22.8 9.1±12.52 23.6-25.8 22.6±3.50 3.4 - 5.2 4.2±0.9 8 0.5-0.8 0.66±1.34 Ulcerat e 7 M 4 - 8 5.8±1.68 25.3 - 26.0 25.5±0.39 20.3 - 21.2 20.2±1.02 0.0 0.0 0.0 0.0 0.6 7 U 9-25 19±6.05 25.1-25.5 25.3±0.15 21.6-22.5 21.9±0.37 0.0 0.0 0.0 0.0 0.5 - 0.8 (0.62±0.11 ) 8 L 3 9-17 13.8±3.63 25.1-26.0 25.6±0.34 21.2-24.2 23.6±0.54 0.0 0.0 0.0 0.0 0.6 - 0.8 (0.68±0.11 ) FR 8 M 14-25 18.2±6.96 24.9-26.4 25.6±0.58 20.9-22.6 21.2±0.88 23.7-26.6 25.1±1.07 20.6-22.9 21.9±1.03 23.7-26.6 25.1±1.07 3.2 - 5.2 4.4±0.7 7 0.6 8 U 3-32 21.8±10.8 25.1-26.3 25.8±0.45 20.1-22.3 21.2±1.07 22.6-26.1 24.6±1.45 20.0-22.6 21.4±1.11 22.6-26.1 24.1±1.45 2.6 - 3.5 2.8±0.6 7 0.5-0.5 0.58±0.05 9 L 4 11-39 27.4±10.57 23.9 - 25.4 (24.7±0.5 5) 19.8 - 22.4 (20.4±2.0 5) 0.0-27.1 21.1±6.81 0.0 - 23.4 18.4±5.3 0 24.2-27.1 25.1±1.81 2.8 - 4.2 3.5±0.6 7 0.5-0.8 0.62±0.11 P or FR 9 M 6-30 21.6±8.61 24.9-26.4 25.5±0.65 20.9-22.6 21.6±0.95 0.0-24.0 14.2±10.73 0.0-20.01 11.0±8.92 21.2-24.0 22.8±1.76 2.8 - 4.4 3.6±0.8 2 0.5-0.8 0.6±1.22 9 U 7-13 9.8±2.89 25.5-26.2 25.7±0.35 21.4-22.8 21.7±1.12 0.0 - 24.6 14.02±10. 00 0.0 - 20.2 11.04±9.0 3 20.2-24.6 21.4±3.17 2.4 - 4.2 3.6±0.4 8 0.5-0.8 0.6±1.34 E L 3 5-13 8.8±3.49 24.7 - 25.4 24.9±0.2 8 20.6-22.8 21.5±1.32 0.0-24.5 10.7±13.34 0.0 - 21.2 10.8±10. 34 21.2-24.5 22.7±1.34 2.0 - 3.4 2.8±0.5 4 0.6-0.8 0.64±0.09 FR or TP E M 8-17 11.8±5.13 24.7-26.5 25.4±0.73 20.6-22.2 21.0±1.23 0.0 - 25.1 14.4±10.8 8 0.0-20.8 11.4±9.14 21.2-24.6 22.2±1.88 2.4 - 4.2 3.2±0.9 8 0.6 E U 5-13 9.2±3.46 24.5-25.8 25.2±0.46 20.5-22.8 22.0±2.66 0.0-24.5 14.2±10.96 0.0 - 22.2 12.4±9.9 3 21.2-23.5 21.9±1.66 2.4 - 4.4 3.4±0.9 7 0.6 N L 3 - 5 4.0±1.00 23.4 - 24.7 23.9±0.46 20.4 - 21.5 20.8±0.72 0.0 0.0 0.0 0.0 0.6 Ulcerat e N M 11-25 18.2±6.09 23.5 - 24.2 24.0±0.2 2 20.5-21.2 20.8±0.44 0.0-23.2 10.2±12.60 0.0-20.4 9.1±11.12 20.4-23.0 21.2±2.02 2.0 - 4.8 3.5±1.1 8 0.5-0.6 0.58±0.04 N U 35-42 38.8±3.27 25.1-26.9 25.9±0.69 20.1-21.9 20.9±0.89 0.0-23.8 11.1±12.46 0.0-20.9 9.1±11.14 20.2-23.6 22.1±1.46 1.8 - 4.8 3.6±1.1 7 0.6 J. Bio. & Env. Sci. 20 23 72 | Taia et al. St No of Ca TPG SphPG PrPG Exine Radius Pore width PAL EAL Sulcus L Sulcus W Thickness Orn S L 5 8-21 17.2±4.18 24.5 - 25.4 24.9±0.3 8 20.5-22.4 21.9±0.58 0.0-24.8 10.8±13.45 0.0-20.6 11.1±9.07 21.6-24.8 22.8±1.45 2.2 - 4.2 3.8±0.4 2 0.6 Ulcerat e S M 38-42 39.6±1.82 24.2-25.2 24.5±0.41 20.2-22.0 21.5±0.41 0.0-26.8 13.6±12.29 0.0-22.2 12.8±8.57 23.2-24.8 23.8±0.39 2.0 - 3.6 2.8±0.7 8 0.6 S U 6-22 15.2±6.16 24.9-25.2 25.0±0.13 20.9-23.2 22.6±1.13 0.0-26.7 20.2±5.45 0.0-21.5 16.7±4.36 22.6-25.7 24.2±1.35 1.8 - 3.6 2.8±0.7 7 0.6 W L 3 14-25 18.8±5.54 24.7-25.4 25.1±0.26 21.6-22.6 21.9±1.06 0.0-22.8 18.0±3.57 0.0-19.2 14.9±5.37 19.8-21.8 21.0±0.17 3.2 - 5.2 4.2±0.9 8 0.6 Ulcerat e W M 10-13 11.4±1.52 24.2-24.6 24.4±0.15 20.4-22.6 21.4±1.15 0.0-22.4 17.6±4.86 0.0-21.0 16.5±4.24 19.6-21.6 20.4±0.86 3.6 - 5.4 4.4±0.9 6 0.5 - 0.6 (0.58±0.0 4) W U 5-9 7.0±1.58 24.4-25.0 24.7±0.26 20.2-22.4 21.2±1.06 0.0-22.4 14.5±7.02 0.0 - 21.0 14.2±6.3 9 19.8-21.6 20.4±1.52 3.2 - 5.2 4.4±0.7 7 0.6 Abbreviations: Anap.=Anaporate, Aper=Aperture, Ca=Catkin, EAL=Equatorial axis length, FR=Faintly rugate, L=Length, No=Number, Orn=Ornamentation, P=Psilate, PAL=Polar axis length, P/E= Polar/Equatorial axis, PrPG=Prolate pollen grain, Sph=Spherical pollen grain, St=Stages, TP= Tectate perforate, TPG=Total pollen grains, RS/P= Ratio between Spherical /Prolate , W=Width, wRSsT=with Rod shape supra tectum. Fig. 18-30. SEM photographs showing the main different characters within the studied stages. Bar=2µm in 19, 21, 22, 24, 26, 27; Bar=5µm in 18, 23, 25, 28; Bar=10µm in20, 29,30. Fig.18 rounded end sulcus, Fig.19 narrow sulcus and rugate exine, Figs.20& 21 faintly rugate exine, Fig.22 rugate exine, Figs.23& 24 rod shapes exinous protrusions, Fig.25 narrow very long sulcus, Fig.26 tectate perforate exine, Figs.27& 30 ulcerate exine, Figs 28 &29 anapore and wide sulcus. Data analyses Most of the investigated pollen pheno-characters showed highly significant differences between the catkin developmental stages (F2 & P2) and in between the different positions (L, M, U) in each catkin stage (from 1-9 and the four directions F1 & P1), except within few characters (Tables 3). Despite the state of differences recorded within the different positions of the catkins (L, M, U) there were highly significant differences (p≤ 0.001) between the different nine stages of the catkin position. J. Bio. & Env. Sci. 20 23 73 | Taia et al. Table 2. Element contents in both spherical and prolate pollen grains at K line, 20 Kv, and 30 second exposure rate (EDX as X-ray exposure by SEM). Spc. Mag. Count Rate Element Mass% Atom% 010 Fitting ratio 0.2058 700X 2141.00 C 53.22±0.30 63.27±0.36 O 38.23±0.60 34.12±0.54 Na 0.19±0.09 0.12±0.05 Mg 0.39±0.07 0.23±0.04 K 3.40±0.11 1.24±0.04 Cu 2.93±0.21 0.66±0.05 Zn 1.65±0.20 0.36±0.04 Total 100.00 100.00 020 Fitting ratio 0.0314 2500X 1851.00 C 60.59±0.22 68.62±0.25 O 34.78±0.42 29.57±0.36 Mg 0.31±0.03 0.17±0.02 P 1.20±0.04 0.53±0.02 S 0.30±0.02 0.13±0.01 K 2.40±0.06 0.83±0.02 Ca 0.42±0.03 0.14±0.01 Total 100.00 100.00 030 Fitting ratio 0.0291 700X 2141.00 C 57.66±0.19 65.64±0.22 O 38.38±0.39 32.80±0.33 Mg 0.35±0.03 0.20±0.01 P 0.93±0.03 0.41±0.02 S 0.18±0.02 0.08±0.01 K 2.5±0.06 0.88±0.02 Total 100.00 100.00 040 Fitting ratio 0.0250 1100X 2369.00 C 55.96±018 63.75±0.20 O 40.92±0.36 35.00±0.31 Mg 0.28±0.02 0.16±0.01 P 0.93±0.03 0.41±0.01 S 0.15±0.01 0.07±0.01 K 1.76±0.05 0.61±0.02 Total 100.00 100.00 050 Fitting ratio 0.0342 2000X 2081.00 C 58.1±0.20 65.98±0.22 O 38.79±0.39 32.89±0.33 Mg 0.28±0.03 0.16±0.01 P 0.92±0.03 0.40±0.02 S 0.17±0.02 0.07±0.01 K 1.42±0.05 0.49±0.02 Total 100.00 100.00 060 Fitting ratio 0.0266 1300X 2178.00 C 59.25±0.20 67.20±0.22 O 36.46±36 31.05±0.31 Na 0.51±0.04 0.30±0.02 Mg 0.29±0.02 0.16±0.01 P 0.77±0.03 0.34±0.01 S 0.20±0.02 0.09±0.01 Cl 0.95±0.03 0.36±0.01 K 0.93±0.04 0.32±0.01 Ca 0.29±0.02 0.10±0.01 Cu 0.35±0.04 0.07±0.01 Total 100.00 100.00 070 Fitting ratio 0.0343 650X 1815.00 C 54.93±0.22 63.14±0.25 O 40.47±0.42 34.92±036 Na 0.56±0.04 0.34±0.03 Mg 0.25±0.03 0.14±0.02 P 0.94±0.04 0.42±0.02 S 0.23±0.02 0.10±0.01 Cl 0.74±0.03 0.29±0.01 K 1.66±0.05 0.59±0.02 Ca 0.21±0.03 0.07±0.01 Total 100.00 100.00 080 Fitting ratio 0.3520 5000X 2858.00 C 54.77±0.34 66.22±0.41 O 32.21±0.54 29.24±0.49 Na 1.54±0.11 0.98±0.07 Mg 0.62±0.07 0.37±0.04 Cl 2.44±0.08 1.00±0.03 K 1.99±0.09 0.74±0.03 Cu 4.12±0.23 0.94±0.05 Zn 2.31±0.22 0.51±0.05 Total 100.00 100.00 090 Fitting ratio 0.4064 4300X 2644.00 C 54.06±0.35 65.52±0.42 N 0.77±0.50 0.80±0.52 O 33.14±0.61 30.15±0.56 Na 0.20±0.12 0.13±0.07 Mg 0.43±0.08 0.26±0.05 Al 0.95±0.09 0.46±0.05 K 2.05±0.10 0.76±0.04 Cu 5.53±0.29 1.27±0.07 Zn 2.97±0.28 0.66±0.06 Total 100.00 100.00 Table 3. Data analyses (F1, P1, F2, P2) of the different catkin stages (1-9 & E, N, S, N) and microsporangium position (L, M, Up) according to the means and standard deviation in the studied parameters. F: F for One way ANOVA test, pairwise comparison between each 2 groups were done using Post Hoc Test (Tukey), p 1 : p value for comparing between the three studied locations in each stage, p 2 : p value for comparing between the different studied stages. Param → Stages↓ TPG (F1 , P1) No SpPG (F1, P1) R SpPG (F1, P1) No PrPG (F1, P1) L M Up L M Up L M Up L M Up 1 F1 10.008 * , P1 0.003 * F1 11.105*, P1 0.002* F1 2.748, P1 0.104 F1 4.850*, P1 0.029* 2 F1 0.425, P1 0.663 F1 1.596, P1 0.243 F1 4.851*, P1 0.029 F1 106.400*, P1 ˂ 0.001* 3 F1 5.441*, P1 0.021* F1 2.957, P1 0.090 F1 2.140, P1 0.160 F1 8.561*, P1 0.005* 4 F1 1.389, P1 0.287 F1 2.352, P1 0.137 F1 5.260, P1 0.023* F1 0.030, P1 0.971 5 F1 7.506*, P1 0.008* F1 6.000*, P1 0.016* F1 1.043, P1 0.345 F1 2.106, P1 0.164 6 F1 8.745*, P1 0.005* F1 8.745*, P1 0.005* F1 3.933*, P1 0.049* F1 -- , P1 -- 7 F1 8.039*, P1 0.006* F1 9.112*, P1 0.004* F1 5.083*, P1 0.025* F1 2.500, P1 0.124 8 F1 1.032, P1 0.386 F1 0.483, P1 0.629 F1 0.003, P1 0.997 F1 5.346*, P1 0.022* 9 F1 3.639, P1 0.058 F1 0.483, P1 0.629 F1 4.892*, P1 0.028* F1 5.871*, P1 0.017* F2 4.976 * 7.033* 12.023 * 5.464 * 5.415* 15.614 * 3.119* 6.171* 13.188 * 14.771 * 6.033 * 30.082 * P2 ˂ 0.001* ˂ 0.001* 0.009* ˂ 0.001 ˂ 0.001* E F1 1.853, P1 0.199 F12.135, P1 0.161 F1 1.096,P1 0.465 F1 3.573, P1 0.168 N F1 99.186*, P1 ˂ 0.001* F1 75.461*, P1 ˂ 0.001* F1 24.603*, P1 ˂ 0.001* F1 2.533, P1 0.282 S F1 19.853*,P1 ˂ 0.001* F1 24.286*,P1 ˂ 0.001* F1 3.559, P1 0.061 F1 1.439, P1 0.487 W F1 12.462*, P1 0.001* F1 7.175, P1 0.009* F1 11.945*,P1 0.001* F1 6.264*, P1 0.044* F2 6.707 * 54.398 * 70.172 * 6.317* 58.096 * 71.653 * 11.346 * 9.096 * 6.567* 5.927 2.151 5.301 P2 0.004 * ˂0.001* 0.005 * ˂0.001* ˂ 0.001 * 0.001 * 0.004 * 0.115 0.542 0.151