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Pollen morphology of some selected species of the genus Centaurea L. (Asteraceae) from Syria
South African Journal of Botany 125 (2019) 196–201
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Pollen morphology of some selected species of the genus Centaurea L. (Asteraceae) from Syria R. Joujeh a,⁎, S. Zaid a, S. Mona b a b
Department of Plant Biology, Faculty of Science, University of Damascus, Syria Department of Plant Protection, Faculty of Agriculture, University of Aleppo, Syria
a r t i c l e
i n f o
Article history: Received 12 December 2018 Received in revised form 8 June 2019 Accepted 17 July 2019 Available online xxxx Edited by D Honys Keywords: Pollen grains Palynology Centaurea Asteraceae Taxonomy
a b s t r a c t Centaurea genus is one of the most important genera of the family Asteraceae. Palynological study on six species of the genus namely C. iberica, C. virgata, C. verutum, C. hyalolepis, C. solstitialis, C. ammocyanus was carried out. We reported a total of 21 qualitative and quantitative pollen features. Results obtained from the detailed investigation by light microscopy showed that pollen grains of the studied species were similar in shape, size, apertures characters, polarization and symmetry, while they differed in color, exine ornamentation pattern and density of the spines distributed on the exine surface and in quantitative measurements. As a result, we found that some pollen characters proved to be of taxonomic value and are useful for seperating the examined taxa of the genus Centaurea. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction The family Asteraceae is the biggest and the most cosmopolitan of the flowering plants and is probably the most widespread in the Mediterranean (Attard and Cuschieri, 2009). For its multiple properties, Asteraceae is assigned an advanced position in plant systematic (Mbagwu et al., 2008). Several plants in the Asteraceae family are economically important as medicinals, ornamentals and vegetables (Mabel et al., 2014). Centaurea L. is one of the most important genera in the Asteraceae family (Zengin et al., 2016). It is an important member of Cardueae Cass. tribe (Compositae) (Hilpold et al., 2014). It includes large number of species which distributed in SW Asia and the Mediterranean region (Ferrer-Gallegol and Altfnordu, 2016). This genus is formed by annual, biennial or perennial herbaceous plants, less often shrubs (Hilpold et al., 2014). Its medicinal importance as antidiarrhoeal, antidiabetic, antiinflammatory, antibacterial, antipyretic, choleretic, digestive, stomachic, colagog, menstrual, diuretic, hypotensive, astringent has been emphasized by many researchers (Celik et al., 2008). Pollen grains are microscopic structures, that carry the male genetic component gametes of the plants, which produce them (Agashe, 2009). Palynology is the science which studies contemporary and fossil ⁎ Corresponding author at: Department of Plant Biology, Faculty of Science, University of Damascus, Syria. E-mail address: [email protected] (R. Joujeh).
https://doi.org/10.1016/j.sajb.2019.07.040 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
palynomorphs, including spore, pollen, chitinozoans, acritarchs and scolecodonts (Mbagwu et al., 2008). Many taxa in the Asteraceae have characteristic pollen features which can be easily recognized in dispersed fossil pollen (Zavada and Villiers, 2000). Pollen features have great significance in the taxonomy of flowering plants and have been used in establishing relationships in the Asteraceae family (Mabel et al., 2014). The most difficult and important problem of the genus Centaurea is the taxonomy, which has been changed several times over the years (Hayta et al., 2017). Pollen morphology is one of the most significant tools used in a taxonomy of Centaurea (Bancheva et al., 2014). In this context, we aimed in this study to describe in detail the pollen morphological characters of Syrian species of Centaurea, as well as to evaluate the taxonomic value of those data, a seen by light microscopy in the hope of solving some of the problems of taxonomy of this genus. This research is part of our detailed taxonomic study on some species of the genus Centaurea.
2. Materials and methods 2.1. Chemicals Sulfuric acid, ethanol, glycerin, phenol, agar powder, acetic anhydride acid, safranin (Merck, Germany).
R. Joujeh et al. / South African Journal of Botany 125 (2019) 196–201
2.2. Pollen color observations Plant samples were collected from different regions of Syria during our field investigations (Table 1). To observe the pollen color of the studied species, the pollen was collected from open flower anthers and placed on filter papers in the shade away from the air stream, then transferred to glass containers with a perforated cover for ventilation, and kept for one year.
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Table 1 Collection Site of taxa examined. Taxa
Abbreviation
Collection site
C. iberica C. virgata C. verutum C. hyalolepis C. solstitialis C. ammocyanus
CIB CVI CVE CHY CSO CAM
Countryside of Aleppo city Countryside of Damascus city Countryside of Aleppo city Countryside of Homs city Countryside of Damascus city Countryside of Hama city
2.3. Preparation of glycerin gel The glycerin gel is prepared as per to the Kisser method as follows: 175 ml of distilled water was heated up to 50 °C, then 50 g of gel powder was added, the mixture was mixed up several times until dissolved properly. After that, 150 g of glycerin was added, then the mixture was boiled with stirring until it becomes sticky, then 7 g of phenol was added, and the mixture was leaved at room temperature until it cools (Pupuleku et al., 2010).
▪ Prolate P/E × 100 = 134–200 ▪ Perprolate P/E × 100 = N 200
2.6. Pollen size In order to indicate the size of the pollen grain, the length of the larger diameter was adopted. (Erdtman, 1969) has simplified the following six classes of size of pollen grains based on the longest axis:
2.4. Pollen grains preparation for the study with light microscopy Pollen grains were prepared according to the method described by Erdtman, 1969 with few modifications as follows: ▪ Flower buds were selected immediately after collection from the field. Pollen obtained from flower buds' anthers was placed on clean glass slides. Several drops of ethanol (95%) were added and the fatty substances resulting from the addition of alcohol were removed using a blotter. ▪ The grains were then treated with 1–2 drops of Acetolysis mixture (anhydride acetic acid and concentrated sulfuric acid). ▪ The slides were heated on the flame until they get a relatively dark color, then washed with ethanol (70%), cleaned up from the residues of the anthers, and then several drops of the Safranin dye were added, the slides were leaved for 2 min, then a piece of glycerin gel was passed on the dyed slides which then were put on a hot plate to melt the gel. Finally, the slides were covered with glass covers (Pupuleku et al., 2010), and labeled. Acetolyzed pollen grains were studied under binocular light microscope at magnification X1000, they were characterized by the scientific terminology (Erdtman, 1969) and (Nilsson and Muller, 1978). Micrometric ocular lens was used to measure the dimensions of the pollen grains and other measurements. We reported a total of 21 qualitative and quantitative pollen features. Characters were chosen depending on those stated in the literature and our observations of Centaurea species. We offer micrographs of studied pollen grains with descriptions of the features of their structure. 2.5. Pollen shape Erdtman Gunnar proposed specific terms for characterizing pollen shape based on the equatorial view, by calculating the ratio of the polar axis to the equatorial diameter (P: E) as shown in the following equation (Erdtman, 1969): FI = P/E × 100 FI: Form Index P: Polar axis E: Equatorial diameter. Depending on the FI value, the following areas were identified (Agashe, 2009): ▪ ▪ ▪ ▪ ▪
Peroblate P/E × 100 = b50 Oblate P/E × 100 = 70–75 Suboblate P/E × 100 = 89–100 Prolate-spheroidal P/E × 100 = 101–114 Subprolate P/E × 100 = 115–133
▪ ▪ ▪ ▪ ▪ ▪
very small grains b10 μm Small grains 10–25 μm Medium grains 25–50 μm Large grains 50–100 μm Very large grains 100–200 μm Gigantic grains N200 μm
2.7. Pollen apertures The number, position and pattern of the pollen apertures were determined according to NPC-system, which was proposed by (Erdtman and Straka, 1961). 2.8. Statistical analysis Measurements are scored for 30 pollen grains and the values are represented as mean ± SD. The quantitative data was subjected to Hierarchical Cluster Analysis using Statistical Package for Social Science (SPSS) program (version 17). 3. Results 3.1. Pollen color After collecting the pollen grains from the anthers, we noticed that they differed in their colors according to plant species (Table 2 and Fig. 1), and after a year of conservation, the pollen color turned to yellow and yellowish brown. Based on the color of the fresh pollen grains, we categorized the pollen grains into two groups: Group I: pollen grains are white, including C. iberica, C. virgata, and C. ammocyanus. Group II: pollen grains are yellow, including C. verutum, C. hyalolepis, and C. solstitialis.
Table 2 Pollen grains color when they are fresh and after a year of conservation. Taxa
Pollen grain (fresh)
Pollen grain (after year)
CIB CVI CVE CHY CSO CAM
White White Golden yellow Sulfur-yellow Golden yellow Yellowish white
Yellow Yellow Yellowish brown Yellowish brown Yellowish brown Yellow
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Fig. 1. Pollen grains color when they are fresh and after a year of conservation.
Fig. 2. Pollen grains (LM) of species of Centaurea L. (Asteraceae).
R. Joujeh et al. / South African Journal of Botany 125 (2019) 196–201
in all the studied species. Colpi are fissure-like apertures while pores are almost round. Each pollen is characterized by three colpi and three pores, these apertures are arranged equidistantly around the equator of the pollen grains and thus they are of a type trizonocolporate. Under light microscope, it was observed that the colpi are acute at both ends in all species. The pollen grains differed between species in terms of colpus length, colpus width, colpus depth, pore width, distance between colpi. The length of colpus ranged from 21.07 μm in C. verutum, to 28.25 μm in C. iberica, while the width ranged from 3.7 μm in C. verutum, to 8.32 in C. iberica. In terms of the pore width, it ranged from 5.21 μm in C. verutum, to 7.84 μm in C. iberica. We noticed that the width of the colpus was greater than the width of the pore in all species except C. verutum (Table 4). The distance between colpi ranged from 17.36 μm in C. solstitialis, to 20.63 μm, in C. ammocyanus.
Pollen grain dimensions 35 30 25 20 15 10 5 0 CIB
CVI
CVE
polar axis
CHY
CSO
199
CAM
Equatorial diameter
Fig. 3. Comparison of polar axis and equatorial diameter length of studied species.
Table 3 Dimensions, size and shape of pollen grains of studied taxa of Centaurea L. (Asteraceae). Taxa PA (μm)
ED (μm)
PA/ED Shape
Size
CIB
32(33.86 ± 1.6)36
30(31.86 ± 1.3)33
1.06
M
CVI
25.8(26.97 ± 1)28
23.8(25.26 ± 1.5)28
1.07
CVE
25.8(28.16 ± 2.6) 32.3 23.8(25.97 ± 1.8)28
1.08
CHY
26.5(29.49 ± 1.8)31.9
25.4(29.03 ± 1.9)30.8 1.02
CSO
28.8(30.11 ± 1.1)32
26(28.5 ± 1.4)30
1.06
26.2(28.89 ± 1.6)31
1.09
CAM 28.6(31.59 ± 1.7)33
Prolate spheroidal Prolate spheroidal Prolate spheroidal Prolate spheroidal Prolate spheroidal Prolate spheroidal
M M M M M
Pollen grains are measured in microns (1 μm = 0.001 mm). (PA: polar axis; ED: equatorial diameter; M: medium)
3.2. Pollen morphology 3.2.1. Shape and size of the studied pollen In general, results obtained from the detailed investigation by light microscopy showed pollen grains to be isopolar, radially symmetrical. In the equatorial view, pollen grains were almost circular, while in the polar view, they were triangular (Fig. 2). The polar axis ranged from 26.97 μm in C. virgata, to 33.86 μm in C. iberica, the equatorial diameter ranged from 25.26 μm in C. virgata, to 31.86 μm in C. iberica (Fig. 3), with correlation polar axis/equatorial diameter (P/E) ranged from 1.02 in C. hyalolepis to 1.09 μm in C. ammocyanus. Accordingly, FI value ranged from 102 to 109, and thus the pollen grains are described as Prolate-spheroidal. On the basis of the length of the longest axis (polar axis in this study), the pollen grains are classified as medium in size. All of the measurements are shown in (Table 3). 3.2.2. Pollen apertures Generally, the pollen grains of the six species of Centaurea studied are aperturate. Both types of aperture, the colpi and pores are present
3.2.3. Pollen wall The exine sculpture was scabrate in C. iberica, C. virgata, C. hyalolepis, and C. ammocyanus, while it was microechinate in C. verutum and C. solstitialis. Exine was thinnest in C. ammocyanus while it was thickest in C. verutum. Wall thickness ranged from 2.31 μm in C. ammocyanus to 2.96 μm in C. verutum. The distance between spines ranged from 1.61 μm in C. ammocyanus to 3.59 μm in C. verutum. Spine length was shortest in C. verutum and longest in C. iberica. Spine width (at the base) was largest in C. verutum and smallest in C. hyalolepis. On the basis of exine ornamentation observed under LM, we identified two types of pollen grains, type I in which there were dense spines on the exine surface including C. verutum and C. solstitialis, type II in which spines were sparsely distributed on the exine surface including C. iberica, C. virgata, C. hyalolepis and C. ammocyanus. Thus, on the basis of the exine ornamentation, we categorized the pollen grains into two groups: Group I: the exine sculpture was scabrate and the spines were distributed densely on the exine surface (C. iberica, C. virgata, C. hyalolepis, and C. ammocyanus). Group II: the exine sculpture was microechinate and the spines were distributed sparsely on the exine surface (C. verutum and C. solstitialis). All the parameters measured and other characters are shown in (Table 5). Pollen characters of studied Centaurea taxa according to previous studies are listed in (Table 6). We could not find any reference studies of the pollen characters of C. verutum and C. hyalolepis. 3.3. Cluster analysis The taxa studied were subjected to Hierarchical Cluster Analysis using the pollen quantitative data. The result of the cluster analysis is presented in (Fig. 4). There are two major clusters. The first main cluster is delineated into two sub clusters; the first sub cluster comprises of four species namely; C. virgata, C. ammocyanus, C. iberica and C. hyalolepis. C. virgata and C. ammocyanus are closely related and have the highest similarity coefficient level of 0.9978. C. iberica and C. hyalolepis are closely related at similarity coefficient level of 0.997. C. iberica and C. virgata are linked together at coefficient level of 0.9965. The four species in the first sub cluster are linked with C. solstitialis which is in the second
Table 4 Pollen apertures data of studied taxa of Centaurea L. (Asteraceae). Taxa
AP
PW (μm)
CL (μm)
CW (μm)
CL/CW
CD (μm)
CCD (μm)
CIB CVI CVE CHY CSO CAM
TZC TZC TZC TZC TZC TZC
7.12(7.84 ± 1.1)9.8 4.5(5.79 ± 1)7 3.5(5.21 ± 1.6)7.5 5.2(6.54 ± 1.3)8.5 4.5(6.07 ± 1.3)8 6(6.5 ± 0.4)7
26.54(28.25 ± 1.4)30 22.8(23.46 ± 0.6) 24.3 18(21.07 ± 2.7)24.5 22.8(24.71 ± 1.5)26.2 23.71(24 ± 2.9)27 26(27.29 ± 1.4)29
7.21(8.32 ± 1.2)10 5.8(6.73 ± 0.9)8 3.3(3.7 ± 0.4)4.3 6(8.01 ± 1.9)10 6(7.17 ± 1.2)8.7 6.2(7.29 ± 0.7)8
3.4 3.5 5.7 3.1 3.3 3.7
7.31(9.25 ± 1.9)12.42 4.8(5.44 ± 0.5)6 4(4.81 ± 0.7)5.8 5.4(6.46 ± 0.8)7.8 5.5(6.69 ± 1)8 5(5.51 ± 0.5)6.3
14.54(20.61 ± 3.1)24.11 14.8(15.77 ± 0.8)17 15.5(15.94 ± 0.6)16.9 18.7(20.1 ± 1)21.6 14(17.36 ± 1.6)19 19(20.63 ± 1.5)22.4
AP: aperture pattern; TZC: trizonocolporate; PW: pore width; CL: colpus length; CW: colpus width; CD: colpus depth; CCD: colpus-colpus distance.
200
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Table 5 Pollen grains walls data of studied taxa of Centaurea L. (Asteraceae). Taxa
WT (μm)
SL (μm)
SW (μm)
SD (μm)
ES
CIB CVI CVE CHY CSO CAM
2(2.33 ± 0.2)2.7 2(2.37 ± 0.5)3 2(2.96 ± 0.9)4.2 2(2.46 ± 0.4)3 2(2.51 ± 0.4)3 2(2.31 ± 0.3)2.7
0.2(0.32 ± 0.1)0.5 0.25 (0.4 ± 0.1)0.6 1(1.57 ± 0.4)2 0.4(0.7 ± 0.2)1 0.7(1.09 ± 0.3)1.6 0.5(0.36 ± 0.1)0.25
0.8(0.93 ± 0.1)1 1(1.46 ± 0.4)2 2.2(3.17 ± 0.7)4 0.6(0.9 ± 0.2)1.1 1.8(1.96 ± 0.1)2 1(1.24 ± 0.2)1.6
1.7(2.11 ± 0.3)2.5 2(2.19 ± 0.2)2.5 2(3.59 ± 0.8)4.3 1.7(1.9 ± 1.1)2 3(3.4 ± 0.5)4 1.3(1.61 ± 0.2)2
S S ME S ME S
Scabrate: spine length less than 1 μm; Microechinate: spine lenglit at range 1–2 μm (Wagenitz, 1955). WT: wall thickness; SL: spine length; SW: spine width (at the base); SD: spine-spine distance (the distance between two spines peaks); ES: exine sculpture; S: scabrate; ME: microechinate.
Table 6 Pollen characters of studied Centaurea taxa according to previous studies. Sp
C. ammocyanus
C. solstitialis
Ref
El Karemy (2000)
Ozler et al. (2009)
Jafari and Ghanbarian (2007)
Biyiklioğlu et al. (2018)
Jafari and Ghanbarian (2007)
C. virgata Biyiklioğlu et al. (2018)
Biyiklioğlu et al. (2018)
C. iberica
PA ED Shape AP PW CL CW CD CCD WT SL SW SD ES
26.9 20.7 Subprolate Tricolporate – – – – – – – – – Scabrate
40.06 32.69 Subprolate Tricolporate 8.93 35.84 6.79 – – – 0.95 2.04 – Scabrate
28.6–36.0 25.0–30.4 Subprolate Tricolporate – – – – – – – – – –
31.8 25.7 Subprolate Tricolporate 8.6 26 3.7 – – 2.5 – – – Microechinate-perfolate
23.0–27.8 23.0–27.8 Spheroidal Tricolporate – – – – – – – – – –
30.4 28 Prolate spheroidal Tricolporate 9.8 27.2 7.9 – – 2.7 – – – Microechinate-perfolate
34.8 30 Subprolate Tricolporate 12.3 28.9 5.9 – – 2.4 – – – Microechinate
PA: polar axis; ED: equatorial diameter; AP: aperture pattern; PW: pore width; CL: colpus length; CW: colpus width; CD: colpus depth; CCD: colpus-colpus distance; WT: wall thickness; SL: spine length; SW: spine width; SD: spine-spine distance; ES: exine sculpture; Sp: species; Ref: Reference; All measurments are in μm.
sub cluster at similarity coefficient level of 0.996. The second main cluster includes only C. verutum which is distinctly separated from the other species.
4. Discussion Pollen research is helpful to understand the systematic and evolutionary relationships of various groups of flowering plants (Stephen et al., 2017). In the current study, pollen grains were prepared using Acetolysis method, which is the basic method for studying the pollen grains by LM, because it helps to better observe the pollen wall structure (Hesse et al., 2009). The Acetolysis mixture dissolves all the contents of the pollen except the outer wall (Exine), which contains sporopolenin which makes it resistant to the mixture (Agashe, 2009). It has been proved that morphological characters of pollen grains are useful in the systematics of the Asteraceae (Shabestari et al., 2013). Centaurea genus presents nomenclatural and taxonomic problems. Systematics of this genus has changed dramatically over the last two decades (Hilpold et al., 2014). Palynological, karyological, and molecular investigations are very useful in solving these problems (Ozler et al., 2009).
The results indicated that the pollen of the six taxa investigated were similar in shape, size, apertures features (number, position and character), polarization and symmetry, while they differed in color, exine ornamentation pattern and density of the spines which distributed on the exine surface and in quantitative parameters such as the length of the polar axis and equatorial diameter, colpus length, colpus width, colpus depth, pore width, the distance between colpi, wall thickness, spine length, spine width, the distance between spines. The first detailed studies of pollen morphology in this genus were done by Wagenitz (1955) and Avetisjan (1964). In Wagenitz (1955), Centaurea genus was divided into eight groups on the basis of exine structure and sculpturing: Centaurium, Scabiosa, Serratula, Jacea, Dealbata, Montana, Cyanus and Crupina. In Avetisjan (1964) five pollen types were identified as Centaurium, Jacea, Scabiosa, Psephellus and Serratula (Ozler et al., 2009). The results of the current study were consistent with (Wagenitz, 1955). All taxa examined in the current study belong to Jacea type according to Wagenitz classification, which distinguished three types of exine ornamentation (microechinate, echinate, scabrate) within this group. There have been also several previous studies evaluated the pollen morphology of some Centaurea species (Wagenitz and Hellwig, 2000;
Fig. 4. Hierarchical Cluster Analysis of the Centaurea species studied based on quantitative pollen grain data.
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El Karemy, 2000; Jafari and Ghanbarian, 2007; Celik et al., 2008; Ozler et al., 2009; Kaya et al., 2010; Shabestari et al., 2013; Hayta et al., 2017; Biyiklioğlu et al., 2018) none of which were conducted in Syria. Comparison of our results to the results of previous studies which were listed in Table 6, reveals that our results show similarities with previous reports. Our results coincided with previous studies in terms of the pollen apertures features (number, position, and character), this can be explained by the fact that these features are genetic (Hesse et al., 2009). The differences in the dimensions of pollen may be due to the different environmental factors. The present study showed that the characteristics of pollen walls have a significant taxonomic value in distinguishing between the studied species. This finding is consistent with the results of other studies which showed that the characteristics of pollen walls in terms of structure and exine ornamentation pattern are useful in taxonomic studies of this genus (Ozler et al., 2009; Shabestari et al., 2013). The similarity in structure demonstrated interspecies relationships and reasons for these species to be in the same genus. On the other hand, the differences in quantitative parameters and other features presented reasons for these species to be distinctive species. As a result, we found that some pollen characters proved to be of taxonomic value and are useful in determination of the target Centaurea species. 5. Conclusion In this paper we presented detailed quantitative and qualitative data on the pollen morphology of six Syrian Centaurea species, in attempts to solve some of the taxonomic problems in this genus. As a result, we found that some pollen characters proved to be of taxonomic value and are useful for seperating the examined taxa of the genus Centaurea. Further morphological, karyological, and molecular investigations are necessary for solving problems related to the taxonomy of this genus. Declaration of Competing Interest The authors declare that there is no conflict of interest. Acknowledgements The authors are grateful to the Damascus University (Department of Botany) for financial and technical support of this research. References Agashe, S.N., 2009. Pollen and Spores. Published by Science Publishers, Enfield, NH, USA, p. 400. Attard, E., Cuschieri, A., 2009. In vitro immunomodulatory activity of various extracts of Maltese plants from the Asteraceae family. J. Med. Plants Res. 3, 457–461.
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Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Pollen morphology of some selected species of the genus Centaurea L. (Asteraceae) from Syria R. Joujeh a,⁎, S. Zaid a, S. Mona b a b
Department of Plant Biology, Faculty of Science, University of Damascus, Syria Department of Plant Protection, Faculty of Agriculture, University of Aleppo, Syria
a r t i c l e
i n f o
Article history: Received 12 December 2018 Received in revised form 8 June 2019 Accepted 17 July 2019 Available online xxxx Edited by D Honys Keywords: Pollen grains Palynology Centaurea Asteraceae Taxonomy
a b s t r a c t Centaurea genus is one of the most important genera of the family Asteraceae. Palynological study on six species of the genus namely C. iberica, C. virgata, C. verutum, C. hyalolepis, C. solstitialis, C. ammocyanus was carried out. We reported a total of 21 qualitative and quantitative pollen features. Results obtained from the detailed investigation by light microscopy showed that pollen grains of the studied species were similar in shape, size, apertures characters, polarization and symmetry, while they differed in color, exine ornamentation pattern and density of the spines distributed on the exine surface and in quantitative measurements. As a result, we found that some pollen characters proved to be of taxonomic value and are useful for seperating the examined taxa of the genus Centaurea. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction The family Asteraceae is the biggest and the most cosmopolitan of the flowering plants and is probably the most widespread in the Mediterranean (Attard and Cuschieri, 2009). For its multiple properties, Asteraceae is assigned an advanced position in plant systematic (Mbagwu et al., 2008). Several plants in the Asteraceae family are economically important as medicinals, ornamentals and vegetables (Mabel et al., 2014). Centaurea L. is one of the most important genera in the Asteraceae family (Zengin et al., 2016). It is an important member of Cardueae Cass. tribe (Compositae) (Hilpold et al., 2014). It includes large number of species which distributed in SW Asia and the Mediterranean region (Ferrer-Gallegol and Altfnordu, 2016). This genus is formed by annual, biennial or perennial herbaceous plants, less often shrubs (Hilpold et al., 2014). Its medicinal importance as antidiarrhoeal, antidiabetic, antiinflammatory, antibacterial, antipyretic, choleretic, digestive, stomachic, colagog, menstrual, diuretic, hypotensive, astringent has been emphasized by many researchers (Celik et al., 2008). Pollen grains are microscopic structures, that carry the male genetic component gametes of the plants, which produce them (Agashe, 2009). Palynology is the science which studies contemporary and fossil ⁎ Corresponding author at: Department of Plant Biology, Faculty of Science, University of Damascus, Syria. E-mail address: [email protected] (R. Joujeh).
https://doi.org/10.1016/j.sajb.2019.07.040 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
palynomorphs, including spore, pollen, chitinozoans, acritarchs and scolecodonts (Mbagwu et al., 2008). Many taxa in the Asteraceae have characteristic pollen features which can be easily recognized in dispersed fossil pollen (Zavada and Villiers, 2000). Pollen features have great significance in the taxonomy of flowering plants and have been used in establishing relationships in the Asteraceae family (Mabel et al., 2014). The most difficult and important problem of the genus Centaurea is the taxonomy, which has been changed several times over the years (Hayta et al., 2017). Pollen morphology is one of the most significant tools used in a taxonomy of Centaurea (Bancheva et al., 2014). In this context, we aimed in this study to describe in detail the pollen morphological characters of Syrian species of Centaurea, as well as to evaluate the taxonomic value of those data, a seen by light microscopy in the hope of solving some of the problems of taxonomy of this genus. This research is part of our detailed taxonomic study on some species of the genus Centaurea.
2. Materials and methods 2.1. Chemicals Sulfuric acid, ethanol, glycerin, phenol, agar powder, acetic anhydride acid, safranin (Merck, Germany).
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2.2. Pollen color observations Plant samples were collected from different regions of Syria during our field investigations (Table 1). To observe the pollen color of the studied species, the pollen was collected from open flower anthers and placed on filter papers in the shade away from the air stream, then transferred to glass containers with a perforated cover for ventilation, and kept for one year.
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Table 1 Collection Site of taxa examined. Taxa
Abbreviation
Collection site
C. iberica C. virgata C. verutum C. hyalolepis C. solstitialis C. ammocyanus
CIB CVI CVE CHY CSO CAM
Countryside of Aleppo city Countryside of Damascus city Countryside of Aleppo city Countryside of Homs city Countryside of Damascus city Countryside of Hama city
2.3. Preparation of glycerin gel The glycerin gel is prepared as per to the Kisser method as follows: 175 ml of distilled water was heated up to 50 °C, then 50 g of gel powder was added, the mixture was mixed up several times until dissolved properly. After that, 150 g of glycerin was added, then the mixture was boiled with stirring until it becomes sticky, then 7 g of phenol was added, and the mixture was leaved at room temperature until it cools (Pupuleku et al., 2010).
▪ Prolate P/E × 100 = 134–200 ▪ Perprolate P/E × 100 = N 200
2.6. Pollen size In order to indicate the size of the pollen grain, the length of the larger diameter was adopted. (Erdtman, 1969) has simplified the following six classes of size of pollen grains based on the longest axis:
2.4. Pollen grains preparation for the study with light microscopy Pollen grains were prepared according to the method described by Erdtman, 1969 with few modifications as follows: ▪ Flower buds were selected immediately after collection from the field. Pollen obtained from flower buds' anthers was placed on clean glass slides. Several drops of ethanol (95%) were added and the fatty substances resulting from the addition of alcohol were removed using a blotter. ▪ The grains were then treated with 1–2 drops of Acetolysis mixture (anhydride acetic acid and concentrated sulfuric acid). ▪ The slides were heated on the flame until they get a relatively dark color, then washed with ethanol (70%), cleaned up from the residues of the anthers, and then several drops of the Safranin dye were added, the slides were leaved for 2 min, then a piece of glycerin gel was passed on the dyed slides which then were put on a hot plate to melt the gel. Finally, the slides were covered with glass covers (Pupuleku et al., 2010), and labeled. Acetolyzed pollen grains were studied under binocular light microscope at magnification X1000, they were characterized by the scientific terminology (Erdtman, 1969) and (Nilsson and Muller, 1978). Micrometric ocular lens was used to measure the dimensions of the pollen grains and other measurements. We reported a total of 21 qualitative and quantitative pollen features. Characters were chosen depending on those stated in the literature and our observations of Centaurea species. We offer micrographs of studied pollen grains with descriptions of the features of their structure. 2.5. Pollen shape Erdtman Gunnar proposed specific terms for characterizing pollen shape based on the equatorial view, by calculating the ratio of the polar axis to the equatorial diameter (P: E) as shown in the following equation (Erdtman, 1969): FI = P/E × 100 FI: Form Index P: Polar axis E: Equatorial diameter. Depending on the FI value, the following areas were identified (Agashe, 2009): ▪ ▪ ▪ ▪ ▪
Peroblate P/E × 100 = b50 Oblate P/E × 100 = 70–75 Suboblate P/E × 100 = 89–100 Prolate-spheroidal P/E × 100 = 101–114 Subprolate P/E × 100 = 115–133
▪ ▪ ▪ ▪ ▪ ▪
very small grains b10 μm Small grains 10–25 μm Medium grains 25–50 μm Large grains 50–100 μm Very large grains 100–200 μm Gigantic grains N200 μm
2.7. Pollen apertures The number, position and pattern of the pollen apertures were determined according to NPC-system, which was proposed by (Erdtman and Straka, 1961). 2.8. Statistical analysis Measurements are scored for 30 pollen grains and the values are represented as mean ± SD. The quantitative data was subjected to Hierarchical Cluster Analysis using Statistical Package for Social Science (SPSS) program (version 17). 3. Results 3.1. Pollen color After collecting the pollen grains from the anthers, we noticed that they differed in their colors according to plant species (Table 2 and Fig. 1), and after a year of conservation, the pollen color turned to yellow and yellowish brown. Based on the color of the fresh pollen grains, we categorized the pollen grains into two groups: Group I: pollen grains are white, including C. iberica, C. virgata, and C. ammocyanus. Group II: pollen grains are yellow, including C. verutum, C. hyalolepis, and C. solstitialis.
Table 2 Pollen grains color when they are fresh and after a year of conservation. Taxa
Pollen grain (fresh)
Pollen grain (after year)
CIB CVI CVE CHY CSO CAM
White White Golden yellow Sulfur-yellow Golden yellow Yellowish white
Yellow Yellow Yellowish brown Yellowish brown Yellowish brown Yellow
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Fig. 1. Pollen grains color when they are fresh and after a year of conservation.
Fig. 2. Pollen grains (LM) of species of Centaurea L. (Asteraceae).
R. Joujeh et al. / South African Journal of Botany 125 (2019) 196–201
in all the studied species. Colpi are fissure-like apertures while pores are almost round. Each pollen is characterized by three colpi and three pores, these apertures are arranged equidistantly around the equator of the pollen grains and thus they are of a type trizonocolporate. Under light microscope, it was observed that the colpi are acute at both ends in all species. The pollen grains differed between species in terms of colpus length, colpus width, colpus depth, pore width, distance between colpi. The length of colpus ranged from 21.07 μm in C. verutum, to 28.25 μm in C. iberica, while the width ranged from 3.7 μm in C. verutum, to 8.32 in C. iberica. In terms of the pore width, it ranged from 5.21 μm in C. verutum, to 7.84 μm in C. iberica. We noticed that the width of the colpus was greater than the width of the pore in all species except C. verutum (Table 4). The distance between colpi ranged from 17.36 μm in C. solstitialis, to 20.63 μm, in C. ammocyanus.
Pollen grain dimensions 35 30 25 20 15 10 5 0 CIB
CVI
CVE
polar axis
CHY
CSO
199
CAM
Equatorial diameter
Fig. 3. Comparison of polar axis and equatorial diameter length of studied species.
Table 3 Dimensions, size and shape of pollen grains of studied taxa of Centaurea L. (Asteraceae). Taxa PA (μm)
ED (μm)
PA/ED Shape
Size
CIB
32(33.86 ± 1.6)36
30(31.86 ± 1.3)33
1.06
M
CVI
25.8(26.97 ± 1)28
23.8(25.26 ± 1.5)28
1.07
CVE
25.8(28.16 ± 2.6) 32.3 23.8(25.97 ± 1.8)28
1.08
CHY
26.5(29.49 ± 1.8)31.9
25.4(29.03 ± 1.9)30.8 1.02
CSO
28.8(30.11 ± 1.1)32
26(28.5 ± 1.4)30
1.06
26.2(28.89 ± 1.6)31
1.09
CAM 28.6(31.59 ± 1.7)33
Prolate spheroidal Prolate spheroidal Prolate spheroidal Prolate spheroidal Prolate spheroidal Prolate spheroidal
M M M M M
Pollen grains are measured in microns (1 μm = 0.001 mm). (PA: polar axis; ED: equatorial diameter; M: medium)
3.2. Pollen morphology 3.2.1. Shape and size of the studied pollen In general, results obtained from the detailed investigation by light microscopy showed pollen grains to be isopolar, radially symmetrical. In the equatorial view, pollen grains were almost circular, while in the polar view, they were triangular (Fig. 2). The polar axis ranged from 26.97 μm in C. virgata, to 33.86 μm in C. iberica, the equatorial diameter ranged from 25.26 μm in C. virgata, to 31.86 μm in C. iberica (Fig. 3), with correlation polar axis/equatorial diameter (P/E) ranged from 1.02 in C. hyalolepis to 1.09 μm in C. ammocyanus. Accordingly, FI value ranged from 102 to 109, and thus the pollen grains are described as Prolate-spheroidal. On the basis of the length of the longest axis (polar axis in this study), the pollen grains are classified as medium in size. All of the measurements are shown in (Table 3). 3.2.2. Pollen apertures Generally, the pollen grains of the six species of Centaurea studied are aperturate. Both types of aperture, the colpi and pores are present
3.2.3. Pollen wall The exine sculpture was scabrate in C. iberica, C. virgata, C. hyalolepis, and C. ammocyanus, while it was microechinate in C. verutum and C. solstitialis. Exine was thinnest in C. ammocyanus while it was thickest in C. verutum. Wall thickness ranged from 2.31 μm in C. ammocyanus to 2.96 μm in C. verutum. The distance between spines ranged from 1.61 μm in C. ammocyanus to 3.59 μm in C. verutum. Spine length was shortest in C. verutum and longest in C. iberica. Spine width (at the base) was largest in C. verutum and smallest in C. hyalolepis. On the basis of exine ornamentation observed under LM, we identified two types of pollen grains, type I in which there were dense spines on the exine surface including C. verutum and C. solstitialis, type II in which spines were sparsely distributed on the exine surface including C. iberica, C. virgata, C. hyalolepis and C. ammocyanus. Thus, on the basis of the exine ornamentation, we categorized the pollen grains into two groups: Group I: the exine sculpture was scabrate and the spines were distributed densely on the exine surface (C. iberica, C. virgata, C. hyalolepis, and C. ammocyanus). Group II: the exine sculpture was microechinate and the spines were distributed sparsely on the exine surface (C. verutum and C. solstitialis). All the parameters measured and other characters are shown in (Table 5). Pollen characters of studied Centaurea taxa according to previous studies are listed in (Table 6). We could not find any reference studies of the pollen characters of C. verutum and C. hyalolepis. 3.3. Cluster analysis The taxa studied were subjected to Hierarchical Cluster Analysis using the pollen quantitative data. The result of the cluster analysis is presented in (Fig. 4). There are two major clusters. The first main cluster is delineated into two sub clusters; the first sub cluster comprises of four species namely; C. virgata, C. ammocyanus, C. iberica and C. hyalolepis. C. virgata and C. ammocyanus are closely related and have the highest similarity coefficient level of 0.9978. C. iberica and C. hyalolepis are closely related at similarity coefficient level of 0.997. C. iberica and C. virgata are linked together at coefficient level of 0.9965. The four species in the first sub cluster are linked with C. solstitialis which is in the second
Table 4 Pollen apertures data of studied taxa of Centaurea L. (Asteraceae). Taxa
AP
PW (μm)
CL (μm)
CW (μm)
CL/CW
CD (μm)
CCD (μm)
CIB CVI CVE CHY CSO CAM
TZC TZC TZC TZC TZC TZC
7.12(7.84 ± 1.1)9.8 4.5(5.79 ± 1)7 3.5(5.21 ± 1.6)7.5 5.2(6.54 ± 1.3)8.5 4.5(6.07 ± 1.3)8 6(6.5 ± 0.4)7
26.54(28.25 ± 1.4)30 22.8(23.46 ± 0.6) 24.3 18(21.07 ± 2.7)24.5 22.8(24.71 ± 1.5)26.2 23.71(24 ± 2.9)27 26(27.29 ± 1.4)29
7.21(8.32 ± 1.2)10 5.8(6.73 ± 0.9)8 3.3(3.7 ± 0.4)4.3 6(8.01 ± 1.9)10 6(7.17 ± 1.2)8.7 6.2(7.29 ± 0.7)8
3.4 3.5 5.7 3.1 3.3 3.7
7.31(9.25 ± 1.9)12.42 4.8(5.44 ± 0.5)6 4(4.81 ± 0.7)5.8 5.4(6.46 ± 0.8)7.8 5.5(6.69 ± 1)8 5(5.51 ± 0.5)6.3
14.54(20.61 ± 3.1)24.11 14.8(15.77 ± 0.8)17 15.5(15.94 ± 0.6)16.9 18.7(20.1 ± 1)21.6 14(17.36 ± 1.6)19 19(20.63 ± 1.5)22.4
AP: aperture pattern; TZC: trizonocolporate; PW: pore width; CL: colpus length; CW: colpus width; CD: colpus depth; CCD: colpus-colpus distance.
200
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Table 5 Pollen grains walls data of studied taxa of Centaurea L. (Asteraceae). Taxa
WT (μm)
SL (μm)
SW (μm)
SD (μm)
ES
CIB CVI CVE CHY CSO CAM
2(2.33 ± 0.2)2.7 2(2.37 ± 0.5)3 2(2.96 ± 0.9)4.2 2(2.46 ± 0.4)3 2(2.51 ± 0.4)3 2(2.31 ± 0.3)2.7
0.2(0.32 ± 0.1)0.5 0.25 (0.4 ± 0.1)0.6 1(1.57 ± 0.4)2 0.4(0.7 ± 0.2)1 0.7(1.09 ± 0.3)1.6 0.5(0.36 ± 0.1)0.25
0.8(0.93 ± 0.1)1 1(1.46 ± 0.4)2 2.2(3.17 ± 0.7)4 0.6(0.9 ± 0.2)1.1 1.8(1.96 ± 0.1)2 1(1.24 ± 0.2)1.6
1.7(2.11 ± 0.3)2.5 2(2.19 ± 0.2)2.5 2(3.59 ± 0.8)4.3 1.7(1.9 ± 1.1)2 3(3.4 ± 0.5)4 1.3(1.61 ± 0.2)2
S S ME S ME S
Scabrate: spine length less than 1 μm; Microechinate: spine lenglit at range 1–2 μm (Wagenitz, 1955). WT: wall thickness; SL: spine length; SW: spine width (at the base); SD: spine-spine distance (the distance between two spines peaks); ES: exine sculpture; S: scabrate; ME: microechinate.
Table 6 Pollen characters of studied Centaurea taxa according to previous studies. Sp
C. ammocyanus
C. solstitialis
Ref
El Karemy (2000)
Ozler et al. (2009)
Jafari and Ghanbarian (2007)
Biyiklioğlu et al. (2018)
Jafari and Ghanbarian (2007)
C. virgata Biyiklioğlu et al. (2018)
Biyiklioğlu et al. (2018)
C. iberica
PA ED Shape AP PW CL CW CD CCD WT SL SW SD ES
26.9 20.7 Subprolate Tricolporate – – – – – – – – – Scabrate
40.06 32.69 Subprolate Tricolporate 8.93 35.84 6.79 – – – 0.95 2.04 – Scabrate
28.6–36.0 25.0–30.4 Subprolate Tricolporate – – – – – – – – – –
31.8 25.7 Subprolate Tricolporate 8.6 26 3.7 – – 2.5 – – – Microechinate-perfolate
23.0–27.8 23.0–27.8 Spheroidal Tricolporate – – – – – – – – – –
30.4 28 Prolate spheroidal Tricolporate 9.8 27.2 7.9 – – 2.7 – – – Microechinate-perfolate
34.8 30 Subprolate Tricolporate 12.3 28.9 5.9 – – 2.4 – – – Microechinate
PA: polar axis; ED: equatorial diameter; AP: aperture pattern; PW: pore width; CL: colpus length; CW: colpus width; CD: colpus depth; CCD: colpus-colpus distance; WT: wall thickness; SL: spine length; SW: spine width; SD: spine-spine distance; ES: exine sculpture; Sp: species; Ref: Reference; All measurments are in μm.
sub cluster at similarity coefficient level of 0.996. The second main cluster includes only C. verutum which is distinctly separated from the other species.
4. Discussion Pollen research is helpful to understand the systematic and evolutionary relationships of various groups of flowering plants (Stephen et al., 2017). In the current study, pollen grains were prepared using Acetolysis method, which is the basic method for studying the pollen grains by LM, because it helps to better observe the pollen wall structure (Hesse et al., 2009). The Acetolysis mixture dissolves all the contents of the pollen except the outer wall (Exine), which contains sporopolenin which makes it resistant to the mixture (Agashe, 2009). It has been proved that morphological characters of pollen grains are useful in the systematics of the Asteraceae (Shabestari et al., 2013). Centaurea genus presents nomenclatural and taxonomic problems. Systematics of this genus has changed dramatically over the last two decades (Hilpold et al., 2014). Palynological, karyological, and molecular investigations are very useful in solving these problems (Ozler et al., 2009).
The results indicated that the pollen of the six taxa investigated were similar in shape, size, apertures features (number, position and character), polarization and symmetry, while they differed in color, exine ornamentation pattern and density of the spines which distributed on the exine surface and in quantitative parameters such as the length of the polar axis and equatorial diameter, colpus length, colpus width, colpus depth, pore width, the distance between colpi, wall thickness, spine length, spine width, the distance between spines. The first detailed studies of pollen morphology in this genus were done by Wagenitz (1955) and Avetisjan (1964). In Wagenitz (1955), Centaurea genus was divided into eight groups on the basis of exine structure and sculpturing: Centaurium, Scabiosa, Serratula, Jacea, Dealbata, Montana, Cyanus and Crupina. In Avetisjan (1964) five pollen types were identified as Centaurium, Jacea, Scabiosa, Psephellus and Serratula (Ozler et al., 2009). The results of the current study were consistent with (Wagenitz, 1955). All taxa examined in the current study belong to Jacea type according to Wagenitz classification, which distinguished three types of exine ornamentation (microechinate, echinate, scabrate) within this group. There have been also several previous studies evaluated the pollen morphology of some Centaurea species (Wagenitz and Hellwig, 2000;
Fig. 4. Hierarchical Cluster Analysis of the Centaurea species studied based on quantitative pollen grain data.
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El Karemy, 2000; Jafari and Ghanbarian, 2007; Celik et al., 2008; Ozler et al., 2009; Kaya et al., 2010; Shabestari et al., 2013; Hayta et al., 2017; Biyiklioğlu et al., 2018) none of which were conducted in Syria. Comparison of our results to the results of previous studies which were listed in Table 6, reveals that our results show similarities with previous reports. Our results coincided with previous studies in terms of the pollen apertures features (number, position, and character), this can be explained by the fact that these features are genetic (Hesse et al., 2009). The differences in the dimensions of pollen may be due to the different environmental factors. The present study showed that the characteristics of pollen walls have a significant taxonomic value in distinguishing between the studied species. This finding is consistent with the results of other studies which showed that the characteristics of pollen walls in terms of structure and exine ornamentation pattern are useful in taxonomic studies of this genus (Ozler et al., 2009; Shabestari et al., 2013). The similarity in structure demonstrated interspecies relationships and reasons for these species to be in the same genus. On the other hand, the differences in quantitative parameters and other features presented reasons for these species to be distinctive species. As a result, we found that some pollen characters proved to be of taxonomic value and are useful in determination of the target Centaurea species. 5. Conclusion In this paper we presented detailed quantitative and qualitative data on the pollen morphology of six Syrian Centaurea species, in attempts to solve some of the taxonomic problems in this genus. As a result, we found that some pollen characters proved to be of taxonomic value and are useful for seperating the examined taxa of the genus Centaurea. Further morphological, karyological, and molecular investigations are necessary for solving problems related to the taxonomy of this genus. Declaration of Competing Interest The authors declare that there is no conflict of interest. Acknowledgements The authors are grateful to the Damascus University (Department of Botany) for financial and technical support of this research. References Agashe, S.N., 2009. Pollen and Spores. Published by Science Publishers, Enfield, NH, USA, p. 400. Attard, E., Cuschieri, A., 2009. In vitro immunomodulatory activity of various extracts of Maltese plants from the Asteraceae family. J. Med. Plants Res. 3, 457–461.
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