Bull 325 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy Bull. Iraq nat. Hist. Mus. (2023) 17 (3): 325-347. https://doi.org/10.26842/binhm.7.2023.17.3.0325 ORIGINAL ARTICLE PALYNOLOGICAL STUDIES FOR SOME CULTIVATED SPECIES OF PINUS L., 1753 (PINALES, PINACEAE) IN EGYPT Asmaa Khamis* and Rim Hamdy **♦ *Department of Botany, Faculty of Science, Fayoum University, Fayoum, Egypt. **Department of Botany, Faculty of Science, The Herbarium, Cairo University, Giza, Egypt. ♦ Corresponding author: rhamdy@sci.cu.edu.eg Recived Date: 05 October 2022, Accepted Date 28 December 2022, Published Date:20 June 2023 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT Pollen grains of the five cultivated species of Pinus L., 1753 from Subsect. Pinaster (Order Pinales, Family Pinaceae) were collected from the Orman Botanic Gardens at Giza in addition to the herbarium specimens. They were examined by light and scanning electron microscopy to detect the taxonomic value of their pollen characteristics. The pollen grains were bisaccate. An artificial key constructed according to the morphology of pollen grains recognizes the five species that belong to Pinus: P. pinea Linnaeus, 1753; P. canariensis Smith, 1828; P. halepensis Miller, 1768; P. roxburghii Sargent, 1897; and P. brutia Tenore, 1811. The differential items included the presence or absence of apertures, e.g. the monosulcate colpus that presents in P. pinea and P. brutia; pollen shape without sacci that could be perprolate as in P. pinea or prolate as in the remaining species; pollen shape (outlined with sacci) in polar view that could be haploxylonoid as in P. pinea and P. roxburghii or diploxylonoid as in the remaining species; in addition to cappa and sacci exine sculpture. A dendrogram from the Community Analysis Package statistical program for data analysis supported the separation of five species of Pinus in Egypt and showed that P. canariensis and P. halepensis were closely related, as well as P. brutia and P. roxburghii. The cluster separated P. pinea into a separate group, but it was more closely related to P. canariensis and P. halepensis. The cluster tree was illustrated, visualized, and confirmed by a heat map based on the R programming language for effective manipulation of the data. Keywords: Cultivated trees, Data analysis, Egypt, Pinus, Pollen morphology. INTRODUCTION The family Pinaceae Spreng. ex F. Rudolphi (1830) belongs to the order Pinales Gorozh (1904), which included 200 species grouped under 11 genera and distributed in the Northern Hemisphere (Farjon, 2001). Pinus species are distributed over North Africa, Asia, Europe, and North America (Shu, 1999). Pinus trees dominate Northern Hemisphere forests, including boreal, subalpine, temperate, tropical, and arid woodlands (Richardson and Rundel, 1998). They are sources of economically important wood, paper, resins, and charcoal, and their seeds are used as food as well as for ornamentation (Le Maitre, 1998). BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Iraq Natural History Research Center & Museum, University of Baghdad https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home Copyright © Bulletin of the Iraq Natural History Museum Online ISSN: 2311-9799-Print ISSN: 1017-8678 https://doi.org/10.26842/binhm.7.2023.17.3.0325 https://orcid.org/0000-0001-5043-8711 https://orcid.org/0000-0001-7777-693X mailto:rhamdy@sci.cu.edu.eg https://creativecommons.org/licenses/by/4.0/ https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home 326 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species Large winter buds with numerous scales. Leaves are needle-like, straight or twisted, with persistent or deciduous membranous sheaths at the base (Shu, 1999). Genus Pinus has two subgenera, [Subgenus Pinus L. (1753) and Subgenus Strobus (D. Don) Lemmon (1895)] as classified by Gernandt et al. (2005). In turn, the subgenus Pinus, ‘hard pines’ has two sections [sect. Pinus L. (1753) and sect. Trifoliae Duhamel (1755)]. The section Pinus is comprised of two subsections: Subsect. Pinus L. (1753), and subsect. Pinaster Loudon (1838); the latter subsection includes the five studied Pinus species: P. brutia, P. canariensis, P. halepensis, P. pinea, and P. roxburghii. The section Trifoliae comprises three subsections [subsect. Contortae Little and Critchfield (1969), subsect. Australes Loudon (1838), and subsect. Ponderosae Loudon (1838)]. The subgenus Strobus, ‘soft pines’ has two sections: [Sect. Quinquefoliae Duhamel (1755), including three subsections: Subsect. Strobui Loudon (1838), Subsect. Krempfianae Little and Critchfield (1969), and subsect. Gerardianae Loudon (1838), and sect. Parrya Mayr (1890), with three subsections: namely, Balfourianae Engelm. (1880), Cembroides Engelm. (1880), and Nelsonianae Burgh (1973). Farjon (2001) recognized 109 species in the genus Pinus, while Price et al. (1998) proposed 111 species in two subgenera, four sections, and 17 subsections based on cross- ability, secondary metabolites, protein, DNA, morphological, anatomical, and cytological studies. Pines are trees or rarely shrubs, evergreen, with regularly whorled branches; branchlets are strongly dimorphic: long branchlets bear scale-like leaves; short branchlets are persistent or deciduous and bear acicular leaves in fascicles of 2-5.Male cones are axillary, upright, densely clustered at the base of short shoots, sessile, small, soft, cylindrical, oblong, or ovoid, up to 15 mm long; female cones are terminal or sub-terminal, woody, and collapse as a whole; scales are cuneate, thickened at the apex, and frequently extended; seeds are winged or not (Shu, 1999). Pollen morphology such as size, shape, exine thickness, and sculpture pattern are very important characteristics and can be used for identification and differentiation between related species (Goda and Gabr, 2018). Pollen identification is widely used in the reconstruction of vegetation, past climates, and plant biodiversity. Studies concerning pollen structure, size, and form are key issues in basic sciences such as plant taxonomy and evolution (Dafni et al., 2000). According to Okwulehie and Okoli (1999), pollen morphology differences can not only reclassify the investigated taxa, but also their morphological similarities can indicate interspecific relationships. In general, gymnosperms have great diversity in pollen morphology, which is significant in the classification of their taxa. Pollens of the order Pinales are distinguished by their two sacci and a proximal part called the cap (Khan et al., 2018). Pinaceae is one of the conifer families that is well-known for its saccate pollen as a modified form of pollen grain to facilitate pollination (Tomlinson, 1994). Pinus male cones in clusters have numerous microsporophylls that are considered stamens in angiosperms, within which two microsporangia contain millions of bisaccate microspores, or pollen grains, that are carried 327 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy by the wind during pollination. The recognition of pollen grain characteristics can be used to trace the history of taxa; these have significant taxonomic value (Moore and Webb, 1978). According to Khan et al. (2017), both light microscopy (LM) and scanning electron microscopy (SEM) have significant value in the investigation and identification of pollen grains from various plant taxa. Using SEM, in addition to pollen examination and description by LM, greatly increases resolution and confirms the exine surface morphology, which can be more significant at the species level (Bagnell, 1975; Weir and Thurston, 1977). SEM can be used to identify pollen grains based on external exine features (Jones and Bryant, 2007; Faria et al., 2021), and the internal structure of Pinus pollen bladders (Sivak and Caratini, 1973). Ting (1965) used pollen morphological characteristics to identify 20 taxa within Pinus and other Pinaceae taxa in California. Several authors investigated the pollen morphology of Pinus: Erdtman (1969); Ueno (1978); Iwanami et al. (1988); Song et al. (2012); and Nikolić et al. (2019). Also, the evolutionary study of Liu and Basinger (2000) was carried out on some fossil taxa of Pinaceae (including Pinus) in Canada. Khan et al. (2018) assessed the pollen flora of gymnosperms in Pakistan, and Chropeňová et al. (2016) estimated the Pinus pollen’s sensitivity to air pollution in some sites in the Slovak Mountains. In Egypt, the genus Pinus contributes to 4.1% of the total pollen production of the taxa in the Rosetta district as a result of the work of Taia et al. (2019). As a result, the study of its pollen should be of great interest in Egypt. Palyno-morphological studies of Pinus in Egypt need more elaborated investigation, accordingly, the present study will focus on the pollen grain qualitative and quantitative morphological characters of five Pinus species cultivated in gardens. To provide palynological data to support the taxonomy of the studied Pinus species cultivated in Egypt. MATERIALS AND METHODS Materials data: Herbarium specimens of Pinus from CAI and CAIM were studied, and the freshly collected specimens were compared with authentic specimens by R. Hamdy at the Herbarium of Cairo University. The new collections of the five species from the Orman Botanic Garden east of Cairo University at Giza, Egypt, were done to provide materials for pollen studies. A total of 30 pollen grains were taken from the third pollen sacs from the apex of male strobili for each species and were examined by both light and scanning electron microscopy. The pollen specimens have been preserved in the herbarium of Fayoum University (proposed abbreviation "CAIF"), Fayoum Province, Egypt. An artificial key of pollen morphological characters (observed by both LM and SEM) was constructed for the studied species. The microscopic study by Light microscopy (LM): The collected pollen grains were acetolyzed with glycerin according to a modified protocol of pollen extraction by Moore et al. (1991). By using a light microscope, the Carl Zeiss M305973 at x400, the parameters measured are described in Table (1). Examination of the pollen grains was done in polar and 328 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species equatorial views. Light microscopy photos were captured using an Axiocam 208 colour microscope camera, and the investigation was performed by a PrimoStar Carl Zeiss microscope. Table (1): Pollen morphological data of the examined Pinus species [PL1 (corpus length), PL2 (pollen length with sacci), PW1 (corpus width), PW2 (pollen width with sacci), AL (saccus length), AW (saccus width), ET (exine thickness), FL (furrow length), PPS (Pollen shape in polar view), PS= PL1/PW1, CS (cappa sculpture), and SS (succus sculpture)]. Species PL1 (µm) PL2 (µm) PW1(µm) PW2 (µm) AL (µm) AW (µm) ET (µm) FL (µm) P. pinea 29-33 (31) 34-39 (36.5) 12-18 (15) 17-20 (18.5) 15-18 (16.5) 10-14 (12) 1 1-10 (5.5) P. canariensis 30-37 (33.5) 33-42 (37.5) 14-20 (17) 20-25 (22.5) 11-20 (15.5) 15-19 (17) 1 1-8 (4.5) P. halepensis 27-32 (29.5) 34-43 (38.5) 16-26 (21) 22-28 (25) 17-26 (21.5) 15-25 (20) 1-2 (1.5) 5-9 (7) P. roxburghii 23-30 (26.5) 32-39 (35.5) 16-18 (17) 20-24 (22) 14-18 (16) 13-18 (15.5) 1 5-7 (6) P. brutia 16-25 (20.5) 30-36 (33) 9-20 (14.5) 17-24 (20.5) 12-17 (14.5) 12-18 (15) 1 2-10 (6) Table (1, cont.) Species Size PS Aperture Colpus length PPS Sculpture Shape Ratio CS SS P. pinea M e d iu m -s iz e d g ra in s P e rp ro la te 2.06 M o n o su lc a te 15-17 (16) H a p lo x y lo n o id S c a b ra te P e rf o ra te P. canariensis P ro la te 1.97 A b se n t ـــــــــــ D ip lo x y lo n o id G ra n u la te R u g u la te P. halepensis 1.4 ـــــــــــ V e rr u c a te S c a b ra te P. roxburghii 1.55 ـــــــــــ H a p lo x y lo n o id 329 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy P. brutia S m a ll g ra in s 1.41 M o n o su lc a te 10-21 (15.5) D ip lo x y lo n o id The microscopic study by scanning electron microscopy (SEM): Pollen grains were directly dusted onto carbon stubs provided with double-sided tape, then coated with a 150 Å (15 nm) gold layer. The coating step was made by using the JFC-1100E ion sputter device. The sculpturing pattern of pollen grains per taxon was investigated by using the JSM- 5400LV scanning microscope at (x 500-x 10.000). Pollen terminology used: Pollen grains of the studied species were described according to Praglowski and Punt (1973). Pollen data analysis: Pollen morphological data were based on 14 morphological characters and 30 character states that described pollen dimensions, shape, and exine sculpture as qualitative binary characters and quantitative multi-state characters, respectively, and both were used as operational taxonomic units (OTUs) (Tab. 2). Using the Community Analysis Package (CAP) statistical program, an average linkage agglomerative clustering method was used to obtain a classification dendrogram, and a reciprocal averaging (RA) ordination method was used to obtain an ordination plot, as described by Schütze and Silverstein (1997). The "gg heat map" package version 2.1 (Luo, 2021) was used to create a heat map for clustering visualization. Table (2): Pollen morphological characters, character states, and data matrix applied to the numerical analysis of the examined Pinus species. Characters Character states P. pinea P. canariensis P. halepensis P. roxburghii P. brutia Pollen outline in polar view Diploxylonoid 0 1 1 0 1 Haploxylonoid 1 0 0 1 0 Corpus length (µm) >28.2 1 1 1 0 0 <28.2 0 0 0 1 1 Pollen length with sacci (µm) >36.2 1 1 1 0 0 <36.2 0 0 0 1 1 Corpus width (µm) >16.9 0 1 1 1 0 <16.9 1 0 0 0 1 330 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species Pollen Width with sacci (µm) >21.7 0 1 1 1 0 <21.7 1 0 0 0 1 Saccus length (µm) >16.8 0 0 1 0 0 <16.8 1 1 0 1 1 Saccus width (µm) >15.9 0 1 1 0 0 <15.9 1 0 0 1 1 Exine thickness (µm) =1 1 1 0 1 1 1-2 0 0 1 0 0 Furrow length (µm) >5.8 0 0 1 1 1 <5.8 1 1 0 0 0 Equatorial pollen shape Perprolate 1 0 0 0 0 Prolate 0 1 1 1 1 Ratio between the polar axis and the equatorial diameter >1.67 1 1 0 0 0 <1.67 0 0 1 1 1 Cappa sculpture Scabrate 1 0 0 0 0 Granulate 0 1 0 0 0 Verrucate 0 0 1 1 1 Succus sculpture Psilate 1 0 0 0 0 Rugulate 0 1 0 0 0 Scabrate 0 0 1 1 1 Monosulcate aperture Present 1 0 0 0 1 Absent 0 1 1 1 0 RESULTS The pollen grains of the studied Pinus species were heteropolar, bilaterally symmetric, and bisaccate monads; Table (2) summarized the pollen morphological data of the studied species. Pollen's morphological description of each species (Pl. 1-3) Pinus pinea L., 1753: Tree with a rounded crown; reddish-grey bark with deep longitudinally fissured; yellowish shoots; stiff gray-green leaves arranged in bundles of two, 331 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy 10–20 cm long; male cones broad ovoid; female cones up to 15 cm long, sessile. Corpus lengths of 29–33 µm, pollen lengths with sacci of 34-39 µm, corpus widths of 12 –18 µm, pollen widths with sacci of 17–20 µm, saccus lengths of 15–18 µm, saccus widths of 10–14 µm, exine thickness of one µm, and furrow lengths (leptoma) of 1–10 µm. Equatorial pollen with perprolate shape (PL1/PW1 ratio of 2.06). Monosulcate aperture between pollen sacci with a length of 15–17 µm. Pollen haploxylonoid in polar view. Exine coarse and cappa sculpture scabrate. Sacci sculptures perforate. Pinus canariensis C. Sm. ex DC., 1825: Tree; reddish bark deeply fissured; leaves arranged in bundles of 3, consisting of very long, string-like pendant needles, glossy blue-green, up to 30 cm long, drooping; sheath about 2 cm long; male cones conical-elongated, orange-yellow; female cones cylindrical-ovoid, 20 cm long, arranged in clusters of 2–3, conical-elongated, purplish. Pollen grains with a corpus length of 30–37 µm, pollen length with sacci 33–42 µm, corpus width of 14–20 µm, pollen width with sacci of 20–25 µm, saccus length 11–20 µm, saccus width 15–19 µm, exine thickness of one µm and furrow length 1-8 µm. Equatorial pollen with prolate shape (PL1/PW1 ratio of 1.97). Pollen diploxylonoid in polar view. Exine coarse and cappa sculptures granulate. Sacci sculptures regulate. Pinus halepensis Mill., 1768: Tree, reddish-brown bark glabrous, fissured; pale green shoots; yellowish-brown branches; leaves needle-like, flexible, up to 10 cm long, in clusters of two on dwarf shoots within a sheath; male cones in terminal clusters appear in spring, lanceolate; female cones solitary, pendulous on short axillary branches, ovoid to conical, reddish, and up to 12 cm long. Pollen grains with corpus length of 27–32 µm, pollen length with sacci of 34–43 µm, corpus width of 16–26 µm, pollen width with sacci of 22–28 µm, saccus length 17–26 µm, saccus width 15–25 µm, exine thickness 1–2 µm and furrow length 5-9 µm. Equatorial pollen with prolate shape (PL1/PW1 ratio of 1.40). Pollen diploxylonoid in polar view. Exine coarse and cappa sculpture verrucate. Sacci sculpture scabrate. Pinus roxburghii Sargent, 1897: Evergreen tree, up to 20 m high; branches symmetrically whorled; brownish bark thick, deeply fissured into large plates; leaves needle-like, in groups of threes on dwarf shoots. 20–35 cm long (hence the name longifolia), sheath persistent, 1.2- 2.5 cm long; male cones ovoid, about 1.5 cm long, yellowish in dense terminal clusters; female cones solitary, or 2–3 on the short recurved peduncle, ovoid, 10–20 cm long. Pollen grains had corpus lengths of 23–30 µm, pollen lengths of 32–39 µm, corpus widths of 16–18 µm, pollen widths of 20–24 µm, saccus lengths of 14–18 µm, saccus widths of 13–18 µm, exine thickness of one µm, and furrow lengths of 5–7 µm. Equatorial pollen with prolate shape (PL1/PW1 ratio of 1.55). Pollen haploxylonoid in polar view. Exine coarse and cappa sculpture verrucate. Sacci sculpture scabrate. Pinus brutia Ten., 1811: Tree; orange-red bark deeply fissured; shoots straight, slender; leaves arranged in pairs, bright to yellowish green, 10–18 cm long, slender, about 1 mm thick, with a persistent 1–1.5 cm sheath; female cones sessile to subsessile. Pollen grains had corpus lengths of 16–25 µm, pollen lengths of 30–36 µm, corpus widths of 9–20 µm, pollen widths of 17–24 µm, saccus lengths of 12–17 µm, saccus widths of 12–18 µm, exine 332 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species thickness of one µm, and furrow lengths of 2–10 µm. Equatorial pollen with prolate shape (PL1/PW1 ratio of 1.41). Monosulcate aperture between pollen sacci with a length of 10–21 µm. Pollen diploxylonoid in polar view. Exine coarse and cappa sculpture verrucate. Sacci sculpture scabrate. A key to species was made for the studied specimens to differentiate and correlate among them, according to data based on LM and SEM, resulting in the differentiation of five species as follows: Artificial key to Pinus studied species: 1. Aperture monosulcate ……... …...………………………..…............……. 2 1. Aperture absent.…………………………………………………...……… 3 2. Corpus (without sacci) perprolate…………..…………………….. P. pinea 2. Corpus (without sacci) prolate ………….....……………..……... P. brutia 3. Haploxylonoid in polar view …………………….......…....... P. roxburghii 3. Diploxylonoid in polar view ……………..….......…..………………...….. 4 4. Cappa sculpture granulate, while sacci regulate …………... . P. canariensis 4. Cappa sculpture verrucate, while sacci scabrate ……..…...... P. halepensis 333 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy Plate (1): Light microscopy images, show equatorial view (A, C, E, G, I), and polar view (B, D, F, H, J) of the studied Pinus species; A, B: P. pinea; C, D: P. canareinsis, E, F: P. halepensis, (G, H) P. roxburghii, (I, J) P. brutia. 334 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species Plate (2): Scanning electron microscopy images, show equatorial view (EV) and polar view (PV) of the studied Pinus species: P. pinea: A. EV, B. PV; P. canareinsis: C. EV, D. PV; P. halepensis: E. EV, F. PV; P. roxburghii: G. EV, H. PV; P. brutia: I. EV, J. V. 335 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy Plate(3): Scanning electron microscopy images, show cappa exine sculpture (A, C, E, G and I) and sacci exine sculpture (B, D, F, H, J) of the studied Pinus species: A, B: P. pinea; C, D: P. canareinsis; E, F: P. halepensis; G, H: P. roxburghii and I, J: P. brutia. 336 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species The dendrogram based on the pollen morphological data matrix (Diag. 1) was forked into two clusters; the first one represented P. pinea separately, while the second one combined all the remaining species and was branched into two sub-clusters. The first sub-cluster shared both P. canariensis and P. halepensis then P. roxburghii and P. brutia came together in the second sub-cluster according to some mutual characteristics. A heatmap was obtained for cluster confirmation by utilizing the R program. It depicts the relationships between the studied species (Diag. 2). The reciprocal averaging ordination plot (Diag. 3) also confirmed the dendrogram results. Diagram (1): Dendrogram based on pollen morphological data of the studied Pinus species. 337 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy Diagram (2): Heat map visualize the cluster tree based on pollen morphological data of the studied Pinus species. 338 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species Diagram (3): Reciprocal Averaging (RA) ordination plot based on pollen morphological data of the studied Pinus species. DISCUSSION By applying numerical analyses to the scored data, the agglomerative clustering dendrogram based on pollen morphological data (Diag. 1) revealed the separation of two main groups. Pinus pinea comes in a separate group in 19 due to the perprolate shape of its pollen, scabrate cappa sculpture, and perforate succus sculpture, which are diagnostic characteristics in identification and separation; this matches with the study of Heigl (2020a). The second group made up of 4 species, is divided into two subgroups at 16; the first subgroup consists of P. canariensis and P. halepensis, which appear in a separate group at 12, though each of these two species has distinct characteristics from the other, P. canariensis has granulate cappa sculpture and rugulate succus sculpture, while P. halepensis has average succus length >16.8 µm and exine thickness 1-2 µm, they belong to the same subgroup due to diploxylonoid pollen shape, average corpus length > 28.2 µm, average pollen length with sacci > 36.2 µm [this agrees with that of Heigl (2020b) study where pollen length 41-50 µm], corpus width > 16.9 µm, average pollen width with sacci > 21.7 µm [this is smaller than pollen width of Heigl (2020b) study 51-100 µm], average saccus width > 15.9 µm and prolate pollen shape in equatorial pollen view, in this study the pollen shape of P. halepensis disagrees with the oblate shape of Heigl (2020b) study, it may be due to the differences in pollen width. The second subgroup in the second group combines P. roxburghii and P. brutia at 8. They are closely related and sharing some pollen morphological characteristics such as average corpus length < 28.2 µm, average pollen length with sacci < 36.2 µm [that is smaller than those of Heigl (2021) study 41-50 µm], succus length < 16.8 µm, average saccus width < 15.9 µm, exine thickness of 1 µm, average furrow length > 5.8 µm, prolate pollen shape whereas pollen of Heigl (2021) study have oblate shape due to the differences in pollen 339 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy length. In addition to the verrucate cappa and scabrate succus sculptures that match Heigl (2021), the ratio between the polar axis and the equatorial diameter is 1.67. As seen in pollen data dendrogram, the cluster of P. canariensis and P. halepensis is more related to that of P. roxburghii and P. brutia because they assemble more characters than those with P. pinea. They are common in prolate pollen shapes; at the same time, P. canariensis, P. halepensis, and P. roxburghii are joined in average corpus width > 16.9 µm, average pollen width with sacci > 21.7 µm, and their grains without apertures. Also, P. canariensis, P. halepensis, and P. brutia join in their diploxylonoid pollen. P. canariensis, P. roxburghii, and P. brutia share a saccus length of < 16.8 µm and an exine thickness of 1 µm. In addition, P. halepensis, P. roxburghii, and P. brutia join in an average furrow length of > 5.8 µm, a ratio between the polar axis and the equatorial diameter of < 1.67, a verrucate cappa sculpture, and a scabrate succus sculpture. It is noticeable that the subgroup of P. canariensis and P. halepensis is more adjacent to the cluster of P. pinea because they share common characteristics such as an average corpus length of > 28.2 µm, which is dissimilar to that of P. pinea 31-35 µm and P. halepensis 41- 50 µm in the Heigl (2020 a, b) study, and an average pollen length with sacci > 36.2 µm. P. pinea has a furrow length of < 5.8 µm on average and a ratio of the polar axis to the equatorial diameter of more than 1.67. However, P. pinea shows a connection with the subgroup of P. roxburghii and P. brutia but is less adjacent; that is, all have an average saccus width of < 15.9 µm; both P. pinea and P. roxburghii have haploxylonoid pollens in polar view; in addition, P. pinea joints with P. brutia in average corpus width <16.9 µm, average pollen width with sacci < 21.7 µm and monosulcate apertures are present on the pollen distal part of both species. A heatmap was created using the R-program to confirm the clustering of the studied species and to visualise the relationships between the studied species using a colour gradient, where the darker the shade, the higher the quantitative value and the most common qualitative character (Diag. 2). The resultant five species are separated clearly on a reciprocal averaging ordination plot (Diag. 3) according to the presence or absence of the recorded quantitative and qualitative pollen character states and support the outcomes of the clustering dendrogram. P. pinea looks like a separate entity at 18; P. canariensis is separated at 122 but in the same direction as P. halepensis axis, which is separated at 27. P. roxburghii is separated at -69 and is adjacent to P. brutia, which is separated at -99. The region of separation of P. canariensis and P. halepensis is near the region of separation of P. roxburghii and P. brutia. This is owed to the accuracy of the statistical data analysis in using more diagnostic characteristics in the statistical classification and finding an accurate classification of the studied entities. Among all the studied species, the quantitative and qualitative characters are varied and significant in the characterization of the cultivated Pinus species in Egypt by the 340 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species construction of an artificial key. The exine thickness has significance in the recognition of the species 1-2 µm (1.5 µm on average). This differs from that of the studied P. halepensis, P. brutia, and P. roxburghii in Pakistan (0.2-0.5 µm) by Khan et al. (2018). For the same species, the cluster results of the current study can be compared with the results of the plant morphological description cluster of seven Pinus species by Mohamed (2018), the taxonomic relationships among Pinus taxa according to 21 morphological characters were investigated by using the Average Linkage of the SPSS program; two major clusters resulted; P. halepensis comes in one of these major clusters, while P. pinea, P. canariensis, P. roxburghii, and P. brutia form the other one that is similar to Farjon's (1998) classification of Pinus. The two studies are combatable in the grouping of P. canariensis, P. roxburghii, and P. brutia in one major cluster, but disagree on the combination of P. pinea or P. halepensis with them. A recent study revealed that the bisaccate pollen size (PL2 x PW2) is 30-43 x17–28 µm, the corpus size (PL1 x PW1) is 16–37 x 9–26 µm, the saccus size (AL x AW) is 11–26 x10– 25 µm, and the corpus distal part that is called "furrow" has a length of 1–10 µm. The sacci size is smaller than the corpus size. The largest size of bisaccate grains is of P. halepensis (34–43 x 22–28 µm), while the smallest ones are of P. brutia (30–36 x17–24 µm). These are bigger than those in Pakistan (Khan et al. 2018), 22–27 x14–17 µm for pollen of P. halepensis and 24-33 x17–21 µm for pollen of P. brutia. Also, as it is noticeable, the pollen of Pakistani P. brutia is bigger than that of P. halepensis. In the same debate, the Egyptian pollens of P. roxburghii (32–39 x 20–24 µm) are bigger than the Pakistani ones (25–28 x15– 18 µm) in the same study. On one hand, in the equatorial view, only P. pinea is with perprolate corpus, where the ratio between the polar axis and the equatorial diameter of the investigated pollens exceeds two (2.06), whereas all remaining species are prolate where the ratio is in the range of 1.33-2 (1.40 for P. halepensis and 1.9 for P. canariensis). Also, the pollen of P. pinea and P. brutia has an elongated monosulcate aperture (a single germinal colpus) on the distal pole between the two sacci (Pl. 1, 2). Subjected to the current argument that the pollens of the Egyptian P. halepensis, P. roxburghii, and P. brutia are bigger than the Pakistani ones of the same species, this may be due to the habitual differences that affect pollen size. It is expected that the pollen shape should be different and the pollens appear as oblate in shape and the ratio in a range between 0.5-0.75, where the ratio is 0.63, 0.68, and 0.64 for the pollens of the previously mentioned three species, respectively. These proposed ranges of the ratio between the corpus length and the corpus width are according to Praglowski and Punt (1973). On the other hand, the polar pollen shape is haploxylonoid for P. pinea and P. roxburghii. This is due to the more or less continuous outline of the sacci with the outline of the corpus of the examined pollen then the pollen grains appear more or less smooth ellipsoidal form. However, a diploxylonoid outline is observed for P. canariensis, P. halepensis, and P. brutia because of the discontinuity of the outline of the sacci with the outline of the corpus (Pl. 1, 2), thus the pollen grains appear as three distinct more or less oval parts according to 341 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy Praglowski and Punt (1973). Although the light microscopy investigation data provided valuable characteristics that enabled the construction of the artificial key, these data alone are not sufficient for the differentiation and classification of the species, so SEM investigations are helpful in the present study in the pollen identification of the different species. SEM investigations show that the corpus exine sculpture is varied for the studied species. In P. pinea, exine sculpture is scabrate of irregular shapes less than one µm in all directions, while in P. canariensis, granulate with more or less rounded granules. The sculpturing pattern of the sacci is perforated with a smooth surface and rugulate with irregular elements more than one µm long in both species. While, P. roxburghii, P. halepensis, and P. brutia have the same corpus sculpture, viz., verrucate with wart-like elements of a width of more than one µm and there are no constrictions at the base, and the same sacci sculpture (scabrate) as shown in Pl.3. These disagree with the observation of Khan et al. (2018), where their described pollen exine of P. halepensis, P. brutia, and P. roxburghii in Pakistan showed only one pattern (rugulate). In the case of LM examinations, the sculpture patterns appear reticulate for all studied species, which indicates that LM observations are inaccurate in sculpture descriptions and limited in a taxonomy based on palynological data. So, in addition to LM and SEM, 3D imaging microscopy can be important to understand the structures of the studied pollens, as discussed by Shen et al. (2020). Using advanced microscopy techniques might be more helpful for the investigation and identification of pollen morphology such as Fourier Transform Infrared (FTIR) microspectroscopy as used by Zimmermann (2018) in his study on two Norwegian species, P. mugo and P. sylvestris. Also, the investigation of other parameters than the morphological ones, such as the sequencing of DNA products using whole-genome amplification (WGA) can be considered a precious tool for pollen identification at the species level, as in the work of Nakazawa et al. (2018) on some Russian Pinus species. The dissimilarities between the current results and those of the different studies on the same taxa but from different localities or countries may be due to variations in ecological or geographical conditions or both or may be due to the normal variation among the taxa of the same rank. CONCLUSIONS It is possible to conclude that five species were identified, and the closely related species including P. canariensis and P. halepensis, also P. roxburghii and P. brutia are closely related species; depending on that P. pinea and P. brutia could be distinguished by their monosulcate colpus, pollen of P. pinea was easily identified from the other species by its perprolate shape, P. roxburghii was discriminated by prolate haploxylonoid pollen. The SEM examination distinguished accurately among all the examined species, resulting in the identification of the remaining two species as P. canariensis by the granulate cappa sculpture and rugulate saccus sculpture of its pollen grains, while the pollen grains of P. halepensis were of verrucate cappa sculpture and scabrate saccus sculpture. Finally, based on 342 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Palynological studies for some cultivated species the certainty of the statistical analysis results, two clusters were formed to represent the considered species, P. pinea was easily identified and separated from the other species, P. canariensis and P. halepensis were separated into one group, while both P. roxburghii and P. brutia were closely related in another group, but the first group was more related to P. pinea than the second one. This is required to be confirmed by additional taxonomic tools in the future, such as anatomical and molecular studies. CONFLICT OF INTEREST STATEMENT "The authors declare no conflict of interest". LITERATURE CITED Bagnell, C. R. 1975. Species distinction among pollen grains of Abies, Picea, and Pinus in the Rocky Mountain area (a scanning electron microscope study). Review of Palaeobotany and Palynology, 19(3): 203-220. [CrossRef] Chropeňová, M., Gregušková, E. 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Pinus L., 1753 دراسة حبوب اللقاح لبعض أنواع جنس الصنوبر (Pinales, Pinaceaeفي مصر ) أسماء خميس * ريم حمدي و ** * مصر. ،الفيوم ،جامعة الفيوم/كلية العلوم/قسم النبات ** مصر. ،الجيزة ،جامعة القاهرة /كلية العلوم / و امليكروبيولوجي، املعشبة قسم النبات 20/6/2023، تأريخ النشر 28/12/2022القبول ، تأريخ 5/10/2022:م تأريخ االستل الخلصة ، Pinus L., 1753هذه الدراسة خمس وحدات تصنيفية من جنس الصنوبر تضمنت تم تجميع حبوب اللقاح الخاصة بها من حديقة األورمان النباتية بالجيزة، باإلضافة إلى لقيمة التصنيفية لصفات العينات قيد فحص بعض العينات املعشبية لتحديد ا الدراسة. بدراسة الشكل الظاهري لحبوب اللقاح من خالل امليكروسكوب الضوئي وامليكروسكوب املاسح اإللكتروني، تبين أنها أحادية ذات كيسين جانبيين. تحددت خمسة أنواع لجنس الصنوبر بواسطة مفتاح تعريفي اصطناعي لتعريف الوحدات ؛P. canariensis Smith, 1828 ؛P. pinea Linnaeus, 1753 :يد الدراسةالتصنيفية ق P. halepensis Miller, 1768 ؛ P. roxburghii Sargent, 1897 iو P. brutia Tenore, بناًء على دراسة الصفات الشكلية لحبوب اللقاح، والتي تتضمن وجود أو ،(1811 جسم حبة اللقاح بدون األكياس ، P. brutia و P. pineaغياب شق أحادي كما في أو بيضاوي كما في باقي األنواع، P. pineaالجانبية يمكن أن يكون فوق بيضاوي كما في .Pو P. pineaيظهر محيط حبة اللقاح في املنظر القطبي كوحدة واحدة كما في roxburghii نحت أو كوحدتين متراكبتين كما في باقي األنواع، باإلضافة الى شكل الطبقة الخارجية لحبوب اللقاح. من خالل هذه الدراسة تبين أن الصفات الشكلية نجحت في تعريف، فصل، والتقييم التصنيفي للوحدات التصنيفية قيد لحبوب اللقاح الدراسة. كما دعمت الشجرة التصنيفية الناتجة من برنامج التحليل االحصائي 347 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Khamis and Hamdy Community Analysis Package ة أنواع من جنس الصنوبر في مصر، فصل خمس .Pو P. canariensisفأوضحت الشجرة التصنيفية وجود عالقة قوية بين نوعي halepensis أيًضا وجود عالقة قوية بين نوعي ، وP. brutia وP. roxburghii . جاء في مجموعة تصنيفية منفصلة عن باقي املجموعات، ولكنه في نفس P. pineaوعالن . من أجل معالجة P. halepensisو P. canariensis بالنوعين ت ذو ارتباط الوق خريطة حرارية أفضل للبيانات تم ايضاح وتأكيد الشجرة التصنيفية عن طريق عمل للتحليل االحصائي. 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