Abstract: Pollen morphology of 13 species of genus’ Astragalus distributed in Saudi Arabia was studied with light and electron microscopes. Pollen is generally 3-zonocolporate, perprolate, prolate, subprolate or prolate-spheroidal. Polar axis is ranged from 12.80 to 21.73 μm, while the equatorial axis varies between 24.52 to 37.22 μm. Pollen is trilobulate or triangular in polar outline, elliptic or compressed ovate in equatorial outline. Sculpturing is micro-reticulate, reticulate or rarely perforate in equatorial view (with irregular muri) and psilate, perforate or seldom scabrate (with irregular or circular perforations) in polar view. Six pollen types were recognized: Astragalus asterias pollen type, A. schimperi pollen type, A. palaestinus pollen type, A. spinosus pollen type, A. corrugatus pollen type and A. sieberi pollen type. Description of each type, a key to investigated species as well as SEM micrographs of pollen types is provided. On the other hand, numerical analysis based on UPGMA clustering, factor analysis and factor loading to pollen data has led to recognize two major clads. The second major clad separated into two branches: the first branch includes two subclads and the second branch comprises three species.
INTRODUCTION
Astragalus L., generally considered to be the largest genus in the Angiosperms, encompasses more than 2500 annual and perennial species (Podlech, 1986; Maassoumi, 2005; Lock et al., 1991; Zarre and Podlech, 1997; Maassoumi, 1998; Podlech, 1999, 2010; Podlech et al., 2001; Lock and Schrire, 2005; Zarre et al., 2008). The majority of species are found in the temperate, semiarid and arid continental regions of south-western and central Asia, western North America and along the Andes and Patagonia in South America (Sanderson and Wojciechowski, 1996; Zarre-Mobarakeh, 2000). Moreover, many Astragalus species are distributed in the Mediterranean climatic regions along the Pacific coasts of North and South America and in southern Europe and northern Africa (Lock et al., 1991; Yakovlev et al., 1996; Maassoumi, 1998).
In Saudi Arabia, the genus’ Astragalus is one of the most important genera represented in the flora of the country. About 29 species have been recorded (Collenette, 1999; Chaudhary, 2001). The northern regions of the kingdom with its 25000 km2 shares alone 13 species. Although, there are many systematic studies on the Astragalus species (Osaloo et al., 2003; Pirani et al., 2006; Podlech, 1998; Sanderson and Doyle, 1993; Scherson et al., 2008), some taxonomic problems concerning this genus have not been resolved yet (Wojciechowski et al., 1999; Karamali et al., 2007; Khodaei et al., 2007). The pollen characters of Astragalus have yet to be studied in detail (Tewari and Nair, 1979; Perveen and Qaisar, 1998; Simons and Chinnappa, 2004; Akan et al., 2005; Ekici et al., 2005; Dane et al., 2007; Oskouian et al., 2007; Pinar et al., 2009; Ceter et al., 2013). In this study, the objective is to examine the pollen morphology of the genus in the northern region and to test its taxonomic value. A cladistic analysis (UPGMA clustering, factor analysis and factor loading) based on pollen characters data will help understand better the relationships between species within the genus. To the best of our knowledge, this is the first comprehensive pollen study of this section. The pollen grains of 13 Saudi Astragalus species are examined in details. Then, adequate keys according to pollen characters are established for an easy discrimination of the investigated species. Finally, the specimens from the different studied species of the Astragalus genus are deposited in the herbarium of Arar Science College at the Northern Borders University for conservation purposes.
MATERIALS AND METHODS
Pollen morphology: Thirteen species out from 29 members of the genus’ Astragalus in Saudi Arabia are the subject of the present study. The studied species are arranged alphabetically to facilitate consultation (Table 1). For each species, the valid scientific name is given followed by the citation of the authority and the date of publication. Synonymy is at a minimum to avoid complications. For full synonymy of the species in Collenette (1999), Cope (1985), Migahid (1996) and Chaudhary (2001).
The planting materials (hermaphrodite flowers) are collected from the wild between March and June 2013. The prospected area covers the northern border region with focusing on wadi Arar. The Astragalus’s collection of Herbarium of Range and Animal Development Research Center-Al Jouf has been used as a collection reference, with the amiably cooperation of Al-Hassan (2006).
Specimens of different species are deposited in the herbarium of the college of science, Arar (Northern Border University, Arar, Kingdom of Saudi Arabia).
Pollen materials (buds or anthers) were removed from dry flowers. The studied species are identified according to Collenette (1999), Cope (1985), Migahid (1996) and Chaudhary (2001). Pollen slides were prepared using the technique of Woodehouse method (Wodehouse, 1935). Prepared samples were then examined with Wolfe Digital TM CVM microscope. The measurements were based, at least, on 20 pollen grains for each species. The values of the measurements are presented as a mean value with the maximum and the minimum put between parentheses. For Scanning Electron Microscopy (SEM), dry pollen grains were mounted on stubs and coated with gold (Buyukkartal et al., 2013; Bona, 2013). Pollen grains were examined with a JSM T200 of Electron Microscopy Unit, Assuit University, Assuit, Egypt. The terminology and main morphological concepts are based on Faegri and Iversen (1989), Punt et al. (1994, 2007), Guner et al. (2011) and Akyalcin et al. (2011). The class of pollen shape are subdivided according to the value of the ratio between Polar axis and Equatorial diameter (P/E) as described by Erdtman’s system (Erdtman, 1969) (Table 3, 4).
Numerical analysis: A total of 19 characters were measured for each species, comprising 12 morphometrical (quantitative) and 7 morphological (qualitative) characters. Some character’s observations were omitted and hence they were coded as missing data (-0.999). The 13 species were clustered based on phenotypic traits the scales portray a dissimilarity index calculated using the Euclidean distance coefficient and the dendrogram was developed using UPGMA (unweighted pair group method using arithmetic averages) clustering procedures “according to Seberg et al. (1991) and Sokal and Michener (1958). Factor analysis and factor loading were also applied. All calculations were made using the STATISTICA software (STATISTICA 5.0).
RESULTS
A careful examination of the available pollen material of the 13 species studied belonging to the genus Astragalus revealed the presence of six pollen types (Table 2), which can be distinguished through the following key:
Keys to the pollen types:
| • | Pollen sculpture is reticulate and Muri with the same diameter on all pollen surfaces (A. spinosus type) |
| • | Pollen sculpture is otherwise (2) |
| • | Pollen sculpture is reticulate to psilate (A. schimperi type) |
| • | Pollen sculpture is otherwise (3) |
| • | Pollen sculpture is reticulate to scabrate (A. sieberi type) |
| • | Pollen sculpture is otherwise (4) |
| • | Pollen sculpture is microreticulate to psilate (A. asterias type) |
| • | Pollen sculpture is otherwise (5) |
| • | Pollen sculpture is microreticulate to perforate (A. palastinus type) |
| • | Pollen sculpture is perforate to psilate (A. corrugatys type) |
| Table 1: | A list of the investigated species with their origin and collectors |
| Table 2: | Taxa of Astragalus and their representative pollen types. No. of species = 13 |
| Table 3: | Tabular summary showing the description of LM and SEM samples |
| Col. wid. at Eq.: Colpi width at equator, 1: Microreticulate to psilate, 2: Reticulate to psilate, 3: Microreticulate to perforate, 4: Perforate to psilate, 5: Reticulate, 6: Reticulate to scabrate, I: Muri decrease towards the two pollen poles to form psilate texture and decrease towards pollen apertures to form perforate pattern, II: Muri decrease towards the two pollen poles to form psilate to perforate sculpture and decrease towards pollen apertures to form microreticulate style, III: Muri decrease only towards the two pollen poles to form psilate to perforate texture, IV: Muri decrease towards the two pollen poles and apertures to form perforate form, V: Muri decrease towards the two pollen poles to form psilate sculpture, VI: Muri decrease towards the two pollen poles to from perforate texture and decrease towards apertures margins to form psilate pattern, VII: Muri size the same at all pollen surface, VIII: Muri decrease towards the two pollen poles and apertures to form psilate to perforate texture, IX: Muri decrease towards the two pollen poles and apertures to form scabrate pattern, X: Muri size the same at two pollen poles and decrease at pollen apertures to form perforate texture | |
The following pollen types are recorded among the species of genus’ Astragalus in the flora of Saudi Arabia (Table 2).
Main characters of pollen types:
| • | A. asterias pollen type, SEM (Fig. 1a-c, g-i; Fig. 3d-f; Fig. 5a-c; Table 3, 4) |
Pollen grains are 3-zonocolporate, perprolate (P/E = 2.03-2.56), 13.20 (11.99-13.83)-15.66 (14.09-17.13)x28.80 (27.16-30.58)-37.22 (35.14-39.30) μm, trilobulate in polar outline, with total area 169.54 (154.31-178.58)-225.61 (202.35-249.35) μm, elliptic in equatorial outline, with an area of 370.32 (353.74-386.90)-637.95 (587.78-658.49) μm. Apocolpium diameter is 7.37 (6.84-7.88)-9.70 (9.22-10.32) μm. Colpus is 24.40 (23.27-25.15)-32.00 (31.09-33.11) μm long, 0.88 (0.42-1.36)-1.38 (0.86-1.89) μm wide, narrow or slender at the equator, needle-like, acute or blunt towards the ends. Mesocolpium is 8.40 (6.84-10.59)-11.88 (10.59-13.12) μm wide. Ora is lolongate with elliptic shaped, 5.45 (4.32-6.37)-9.58 (3.19-12.61) μm in diameter and 8.01 (7.86-9.93)-20.61 (14.1-36.03) μm in area. Exine is microreticulate to psilate; Muri decrease towards both the two pollen poles to form psilate texture and towards the pollen apertures to form perforate pattern. Muri decrease only towards the two pollen poles to form psilate to perforate texture or Muri decrease towards the two pollen poles to form psilate sculpture, 0.86 (0.61-1.25)-0.98 (0.86-1.25) μm in diameter.
The following species belong to this type (Table 2):
| • | A. asterias Steven, Bull. Soc. Nat. Moscou 4:267 (1832) |
| • | Syn. A. cruciatussensu Zohary, Fl. Palaest. 2: 59 (1972) |
| • | A. caparinus L. Spp. caparinus L., Sp. Pl., ed. 2, 1071 (1763) |
| • | Syn. A. alexandrines Boiss., Diagn. Pl. Orient., ser. 1, 9: 75 (1849) |
| • | A. lanigerus Desf., Fl. Atlant. 2: 181 (1799) |
| • | A. kahiricus DC., Prodr. 2: 292 (1825). |
| • | Syn. A. longiflorus Delile, Descr. Egypte, Hist. Nat. 356 (1814), non Pallas, Sp. Astragal. 73 (1800-1802), nom illeg |
| • | A. tribuloides Delile var. minutus (Boiss.) Boiss., Fl. Orient. 2: 225 (1872) |
| • | Syn. A. minutus Boiss., Diagn. Pl. Orient., ser. 1, 9: 58 (1849) |
| Table 4: | Tabular summary showing the pollen grains dimensions (µm) |
| P: Polar axis, E: Equatorial diameter, P/E: The ratio of the length of the polar axis (P) to the equatorial diameter (E), Colp. Len: Colpus length, Colp. wid: Colpus width, Ora. diam: Ora diameter, Apo. diam: Apocolpium diameter, Meso. diam: Mesocolpium diameter, µm: Micrometer | |
Key to species of A. asterias pollen type:
| • | Colpus length is 24.40 (23.27-25.15) μm (A. kahiricus) |
| • | Mean of colpus length is 28.19-32.00 (27.58-33.11) μm (2) |
| • | Colpus width is 0.88 (0.42-1.36) μm (A. asterias) |
| • | Mean of colpus width is 1.26-1.38 (1.00-1.89) μm (3) |
| • | Mesocolpium diameter is 11.88 (10.59-13.12) μm (A. caparinus Spp. caparinus) |
| • | Mesocolpium diameter is 9.87 (8.64-9.75) μm (A. tribuloides var. minutus) |
| • | A. schimperi pollen type, SEM (Fig. 1d-f; Fig. 4a-c; Table 3, 4) |
Pollen grains are 3-zonocolporate, prolate orperprolate (P/E = 1.49-2.21), 13.88 (12.57-15.28)-17.40 (16.09-18.77)x25.99 (24.73-27.22)-30.66 (28.43-32.89) μm, trilobulate in polar outline, with an area of 150.49 (135.45-170.49)-228.50 (207.75-243.41) μm, elliptic in equatorial outline, with an area of 356.37 (342.50-370.14)-457.44 (448.08-466.98) μm. Apocolpium diameter is 8.29 (6.29-9.70)-10.24 (9.82-11.47) μm. Colpus is 19.79 (18.05-21.98)-25.68 (24.48-27.47) μm long, 0.74 (0.56-1.24)-1.90 (0.91-2.88) μm wide, slightly wide or slender at the equator, acute or needle-like towards he ends. Mesocolpium is 11.09 (10.38-12.69)-12.12 (10.64-13.54) μm wide. Ora is lolongate or lalongate with elliptic shaped, 4.93 (3.83-5.59)-5.64 (4.31-6.70) μm in diameter and 8.30 (6.71-9.89)-33.60 (30.50-37.76) μm in area. Exine is reticulate to psilate. Muri decrease towards both the two pollen poles to form psilate-perforate sculpture and towards the pollen apertures to form microreticulate style or Muri decrease towards the two pollen poles and apertures to form psilate-perforate texture, 1.26 (0.72-1.71)-1.37 (0.94-1.92) μm in diameter.
The following species belong to this type (Table 2):
| • | A. bombycinus Boiss., Diagn. Pl. Orient., ser. 1, 2: 50 (1843) |
| • | A. schimperi Boiss., Diagn. Pl. Orient., ser. 1, 2: 53 (1843) |
Key to species of A. schimperi pollen type:
| • | Polar view long axis 13.88 (12.57-15.28) μm (A. bombycinus) |
| • | Polar view long axis 17.40 (16.09-18.77) μm (A. schimperi) |
| • | A. palaestinus pollen type, SEM (Fig. 2a-c, g-i; Fig. 3g-i; Table 3, 4) |
Pollen grains are 3-zonocolporate, prolate, subprolate or perprolate (P/E = 1.29-2.61), 12.80 (11.98-13.54)-20.63 (19.50-21.46) x 26.59 (25.68-28.30)-33.41 (31.43-35.39) μm, trilobulate to triangular in polar outline, with an area of 136.04 (123.83-146.06)-273.87 (258.57-287.83) μm, ovate in equatorial outline, with an area of 438.41 (398.47-478.36)-495.82 (486.60-505.03) μm. Apocolpium diameter is 6.53 (4.35-8.71)-10.63 (8.15-12.91) μm. Colpus is 21.46 (16.17-25.04)-28.94 (27.22-30.66) μm long, 1.72 (0.81-2.89)-3.42 (2.18-5.22) μm wide, wide or slightly wide at the equator, acute towards the ends, with scabrate membrane only in A. dactylocarpus Spp. acinaciferus. Mesocolpium is 12.84 (10.66-15.12)-16.45 (14.34-18.64) μm wide. Ora is lalongate with ovate shaped, 6.07 (4.65-7.22)-8.04 (6.97-9.21) μm in diameter and 12.05 (8.05-17.97)-24.91 (21.29-28.50) μm in area. Exine is microreticulate to perforate. Muri decrease towards the two pollen poles and apertures to form perforate form, decrease towards the two pollen poles only to from perforate texture or decrease towards apertures margins to form psilate pattern, 0.87 (0.51-1.35)-0.97 (0.56-1.24) μm in diameter.
The following species belong to this type (Table 2):
| • | A. collenettiae Hedge and Podl., in Bot. Jahrb. Syst. 108, 2/3: 269 (1987) |
| • | A. dactylocarpusSpp. acinaciferus(Boiss.) Eug. Ott, Phanerog. Monogr. 9: 62 (1978). Syn. A. acinaciferus Boiss., Diagn. Pl. Orient., ser. 1, 9: 93 (1849) |
| • | A. palaestinus Eig, J. Bot. 72: 124 (1934) |
Key to species of A. palaestinus pollen type:
| • | The ratio of P/E is 1.29 (A. dactylocarpus spp. acinaciferus) |
| • | The mean ratio of P/E is 1.99-2.61 (2) |
| • | Polar view long axis is 12.80(11.98-13.54) μm (A. collenettiae) |
| • | Polar view long axis is 15.08(14.32-16.69) μm (A. palaestinus) |
| • | A. spinosus pollen type, SEM (Fig. 2a-c, g-i; Fig. 3g-i; Table 3, 4) |
| Fig. 1(a-i): | SEM observations of non acetolyzed pollen grains (SEM×2000-10,000). (a-c) A. asterias: (a) Polar view, (b) Equatorial view, (c) Magnified part of exine. (d-f) A. bombycinus: (d) Obliquepolar view, (e) Equatorial view, (f) Magnified part of exine. (g-i) A. caparinus ssp. caparinus: (g) Polar view, (h) Equatorial view and (i) Magnified part of exine |
| Fig. 2(a-i): | SEM observations of non acetolyzed pollen grains (SEM×2000-10,000). (a-c) A. collenettiae: (a) Polar view, (b) Equatorial view, (c) Magnified part of exine. (d-f) A. corrugatus: (d) Polar view, (e) Equatorial view, (f) Magnified part of exine. (g-i) A. dactylocarpus ssp. acinaciferus: (g) Oblique polar view, (h) Equatorial view and (i) Magnified part of exine |
| Fig. 3(a-i): | SEM observations of non acetolyzed pollen grains (SEM×2000-10,000). (a-c) A. hauarensis: (a) Oblique polar view, (b) Equatorial view, (c) Magnified part of exine. (d-f) A. kahiricus: (d) Polar view, (e) Equatorial view, (f) Magnified part of exine. (g-i) A. palaestinus: (g) Oblique polar view, (h) Equatorial view and (i) Magnified part of exine |
| Fig. 4(a-i): | SEM observations of non acetolyzed pollen grains (SEM×2000-10,000). (a-c) A. schimperi: (a) Polar view, (b) Equatorial view, (c) Magnified part of exine. (d-f) A. sieberi: (d) Oblique polar view, (e) Oblique equatorial view, (f) Magnified part of exine. (g-i) A. spinosus: (g) Oblique polar view, (h) Equatorial view and (i) Magnified part of exine |
| Fig. 5(a-c): | SEM observations of non acetolyzed pollen grains (SEMx2000-10,000). (a-c) A. tribuloides var. minutus: (a) Oblique polar view, (b) Equatorial view and (c) Magnified part of exine |
Pollen grains are 3-zonocolporate, perprolate (P/E = 2.16-2.27), 13.60 (12.29-14.66)-15.94 (14.64-17.16)x30.95 (30.86-31.04)-34.42 (32.17-36.68) μm, trilobulate to triangular in polar outline, with an area of 151.62 (143.53-163.71)-199.25 (174.31-209.79) μm, elliptic in equatorial outline, with an area of 418.57 (414.68-432.46)-472.66 (453.60-487.53) μm. Apocolpium diameter is 5.44 (4.18-6.94)-8.51(7.32-8.70) μm. Colpus is 25.68 (24.88-26.08)-31.44 (30.92-32.05) μm long, 1.42 (1.08-1,71)-1.68 (1.45-1.84) μm wide, narrow or slightly wide at the equator, acute or acuminate towards the ends. Mesocolpium is 10.19 (8.37-11.03)-10.37 (9.09-11.96) μm wide. Ora is lolongate with elliptic shaped, 5.91 (4.31-5.73)-8.19 (7.14-9.24) μm in diameter and 10.40 (8.96-11.85)-13.93 (11.71-14.96) μm in area. Exine is reticulate. Muri diameter is the same on all of the pollen surface or their diameter is the same at two pollen poles only and decrease towards pollen apertures to form perforate texture, 1.34 (0.99-1.87)-1.44 (0.83-1.98) μm in diameter.
The following species belong to this type (Table 2):
| • | A. hauarensisBoiss., Diagn. Pl. Orient., ser. 1, 9: 63 (1849) |
| • | Syn. A. gyzensis Buhge, Mém. Acad. Sci. Pétersb. 11 (16): 14 (1868) |
| • | A. spinosus (Forssk.) Muschl., Verh. Bot. Vereins Prov. Brandenb. 49: 98 (1907) |
| • | Syn. Colutea spinosa Forssk., Fl. Aegypt.-Arab. 131 (1775) |
| • | A. forsskaolii Boiss., Diagn Pl. Orient., ser. 1, 9: 101 (1849), nom. illeg |
Key to species of A. spinosus pollen type:
| • | Colpus length is 25.68 (24.88-26.08) μm (A. hauarensis) |
| • | Colpus length is 31.44 (30.92-32.05) μm (A. spinosus) |
| • | A. corrugatus pollen type, SEM (Fig. 2d-f; Table 3, 4) |
Pollen grains are 3-zonocolporate, perprolate (P/E = 2.34), 13.40 (11.10-14.89)x13.40 (11.10-14.89) μm, triangular in polar outline, with an area of 154.17 (146.38-163.04) μm, ovate in equatorial outline, with an area of 461.21 (458.75-464.66) μm. Apocolpium diameter is 9.18 (8.42-9.67) μm. Colpus is 25.61 (24.94-25.99) μm long, 1.91 (1.39-2.65) μm wide, it is slightly wide at the equator and blunt towards the ends. Mesocolpium is 11.06 (10.54-11.55) μm wide. Ora is lolongate with elliptic shaped, 6.61 (5.38-7.76) μm in diameter and 9.92 (4.61-13.77) μm in area. Exine is perforate to psilate. Muri decrease towards the two pollen poles to form psilate sculpture, 0.38 (0.19-0.54) μm in diameter.
The following taxon belongs to this type (Table 2):
| • | A. corrugatus Bertol., Rar. Ital. Pl. Dec. 3: 33 (1810) |
| • | yn. A. cruciatus Link, Enum. Hort. Berol. Alt. 2: 256 (1822) |
| • | A. sieberi pollen type, SEM (Fig. 2d-f; Table 3, 4) |
Pollen grains are 3-zonocolporate, prolate-spheroidal (P/E = 1.13), 21.73 (20.84-22.64)x24.52 (23.69-25.27) μm, triangular in polar outline, with an area of 334.08 (317.41-351.76) μm, ovate in equatorial outline, with an area of 401.16 (388.16-414.15) μm. Apocolpium diameter is about 7.32 (6.26-8.43) μm. Colpus is 23.24 (21.69-24.06) μm long, 3.61 (2.59-4.11) μm wide, wide at the equator, acute towards the ends, with granulate membrane. Mesocolpium is 17.36 (16.49-18.11) μm wide. Ora is circular in shaped, 7.17 (6.85-7.60) μm in diameter and 26.37 (18.77-36.38) μm in area.
| Table 5: | Factor loadings showed the most intrinsic characters enhanced separations of the studied species |
| Fig. 6: | Phenogram of the 13 studied Astragalus species, clustering with the UPGMA method |
Exine is reticulate to scabrate. Muri decrease towards the two pollen poles and apertures to form scabrate pattern, 1.27 (0.92-1.80) μm in diameter.
The following taxon belongs to this type (Table 2):
| • | A. sieberi DC., Astragalogia 186 (1802) |
| • | Syn. A. leucacanthus Boiss., Diagn. Pl. Orient., ser. 1, 9: 93 (1849) |
On the other hand, Fig. 6 shows the UPGMA cladistic tree of the 13 species of Astragalus depending on 19 main pollen grains characters which discriminate these species into two major clads at 18 dissimilarity distance. The first major clad at 17 dissimilarity distance, comprised only one species of the total number: Astragalus schimperi. The second major clad at 16.5 dissimilarity distance comprised the remaining 12 species. The second major clad separated into two branches. The first branch includes two subclads: (1) A subclad at 13 dissimilarity distance with six species: A. collenettiae, A. corrugatus, A. tribuloides var. minutus, A. hauarensis, A. kahiricus and A. spinosus, (2) A subclad at 12.5 dissimilarity distance with A. asterias, A. caparinus spp. caparinus and A. bombycinus. The second branch of the second major clad comprises three species: A. dactylocarpus spp. acinaciferus, A. palaestinus and A. sieberi at 10.75 dissimilarity distance. Factor analysis using Principal Component Analysis (PCA) showed that the most intrinsic characters enhanced separation of the total species which are pollen shape only of the morphological characters, polar view, equatorial diameter, P/E, colpus length, mesocolpium diameter and polar view total area of the morphometrical characters (Table 5). The characters of separation are of high factor loadings ≥(± 0.7). They are represented by a percentage of the total variation equalling 36.84% for the first factor. however, both the second and the third factors were excluded because there were no characters scored on them (Fig. 7).
| Fig. 7: | Scatter-plot of 13 studied taxa plotted against the first factor by the second factor |
DISCUSSION
In this palynological investigation, an additional perspective on the relations between the different Astragalus species studied was provided. The pollen grains are usually 3-zonocolporate and distinguished according to nature of exine sculpture into six pollen types as the following: The Astragalus asterias pollen type, in which the pollen grains have microreticulate to psilate sculpture, is characteristic of A. asterias, A. caparinus Spp. caparinus, A. kahiricus and A. tribuloides var. minutus. The Astragalus schimperi pollen type, in which the pollen grains have reticulate to psilate sculpture, is characteristic of both A. bombycinus and A. schimperi species. The A. palaestinus pollen type, in which the pollen grains have microreticulate to perforate sculpture, is characteristic of A. collenettiae, A. dactylocarpus Spp. acinaciferus and A. palaestinus species. The A. spinosus pollen type, in which the pollen grains have reticulate sculpture, is characteristic of both A. hauarensis and A. spinosus species. The A. corrugatus pollen type, in which the pollen grains have perforate to psilate sculpture, is characteristic of A. corrugatus species only. Finally, A. sieberi pollen type in which the pollen grains have reticulate to scabrate sculpture, is characteristic of A. sieberi species only (Table 2, 3). Ceter et al. (2013) investigated 15 taxa belonging to the section Hololeuce Bunge of genus Astragalus and demonstrated that, Pollen grains show reticulate, perforate polar sections and perforate, reticulate, microreticulate, perforate-granulate, microreticulate-perforate, microrugulate-perforate, microrugulate-microreticulate, granulate-perforate, microreticulate-granulate at meridional sections. Akan and Aytaç (2014) studied the section Alopecuroidei DC. belonging to genus Astragalus and observed that, the pollen type in the members of this section is subprolate or prolate-spheroid. The pollen is tricolporate and the ornamentation is reticulate. The amb type is semiangular (Akan et al., 2005). Oskouian et al. (2007) examined pollen morphology of 37 Astragalus section Malacothrix and its allies. He observed that, the most common exine surface sculpturing is reticulate, rarely verrucate (only in A. laristanicus of subsect. Bornmuelleriana of sect. Malacothrix). Pollen shape ranges from spheroidal in A. sphaeranthus (sect. Stereothrix) to prolate in the remaining studied species. The current palynological data is of less taxonomic value to distinguish the studied taxa at both species and sectional level.
Pollen size of the studied species ranged between 23-37 μm. The smallest pollen grains are those of A. sieberi 24.52(23.69-25.27) μm and the largest one are those of A. caparinus Spp. caparinus 37.22(35.14-39.30) μm. Pollen size of other species ranged between 25-35 μm (Table 4). Pollen grains which are more or less similar in shape being perprolate, prolate, subprolate or prolate-spheroidal. Colpi are narrow in A. caparinus Spp. caparinus, A. kahiricus, A. spinosus and A. tribuloides var. minutus, wide in A. collenettiae, A. dactylocarpus Spp. acinaciferus and A. sieberi, slender in both A. asterias and A. schimperi and slightly wide in the remaining studied species. Furthermore, colpi ends contribute to differentiate between the species of A. caparinus Spp. caparinus, A. corrugatus and A. tribuloides var. minutus which are characterized by blunt ends. However, both A. asterias and A. schimperi exhibit Needle-like ends. Only A. hauarensis has acuminate ends and the remnant of investigated species have acute ends (Table 3). The granulation of pollen apertures membranes is showed in both A. bombycinus and A. sieberi, scabrate pollen apertures membranes is appeared in A. dactylocarpus Spp. acinaciferus only, while the aperture’s membranes of other species were not cleared.
Moreover, the sculpture at pollen pole could also help in differentiation of A. sieberi, which is characterized by scabrate texture, A. collenettiae, A. dactylocarpus Spp. acinaciferus and A. palaestinus which are characterized by perforate texture, A. hauarensis and A. spinosus which are characterized by reticulate texture from all other remaining species, which possess psilate texture (Table 3). Additionally, the diameter of muri also contributed to differentiating A. bombycinus, A. hauarensis, A. schimperi, A. sieberi and A. spinosus species, which were characterized by large muri with diameter medium 1.26-1.44 μm, from the other remaining investigated species that exhibited small muri with diameter medium 0.38-0.98 μm (Table 3).
On the other hand, a large number of pollen morphology characters were scored and numerical methods (UPGMA and PCA) were applied to study the relationships between thirteen Astragalus species and estimate the level of variation within and between these species. UPGMA gives insight into the degree of similarity between the studied species and whether they form groups (clusters) and give an indication of the level of variation within and between species. PCA reflects which characters are important on the axes and indicates the significant characters on the bases of the highest factor score (Table 5). Therefore, it becomes clear which characters cause the separation between groups and can be useful to distinguish species. Pollen grains showed the most powerful significant characters. Generally, our results arose congruence between the UPGMA clustering and PCA analysis in suggesting two main branches and three subgroups, which included the distribution of thirteen species studied (Fig. 6). Our UPGMA results showed that the A. schimperi is separated in the initial major clad of the cladistic tree and all the remaining 12 species are found in the other major clad. The alternate major clad is separated into two branches. The primary branch includes two subclads. The first subclad includes six species (A. collenettiae, A. corrugatus, A. tribuloides var. minutus, A. hauarensis, A. kahiricus and A. spinosus), while the second subclad comprises three species (A. asterias, A. caparinus spp. caparinus and A. bombycinus). The second branch of the second major clad contains three species (A. dactylocarpus spp. acinaciferus, A. palaestinus and A. sieberi) (Fig. 6). Then, the applied methods of UPGMA and PCA can be used to study the variation among the species in the genus’ Astragalus to determine the relationship between different species. Our results revealed there is a clear separation between A. schimperi and all another investigated species. Moreover, species A. asterias, A. bombycinus and A. caparinus spp. caparinus showed a much closer relationship speciesin the same clad. In addition to species of A. dactylocarpus spp. acinaciferus, A. palaestinus and A. sieberi arises also a much closer relationships being existed in the same sub-branch (Fig. 7).
ACKNOWLEDGMENTS
The authors are grateful to Deanship of Scientific Research, Northern Border University, for its great interest and financial support, which lead to the final successful completion of this research.