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Riosysteras
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ISSN 2519-8513 (Print) ISSN 2520-2529 (Online) Biosyst. Divers., 2020, 28(4), 399-404 doi: 10.15421/012051
Diversity of Sambucus nigra pollen within Slovakia in selected morphological characters by SEM study
V. Horcinova Sedlackova*, O. Grygorieva**, I. Gurnenko**, O. Vergun**
*Institute of Biodiversity Conservation and Biosafety, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic **M M Gryshko National Botanical Garden of Ukraine, National Academy of Sciences, Kyiv, Ukraine
Article info
Received 08.10.2020
Received in revised form 14.11.2020
Accepted 18.11.2020
Institute of Biodiversity Conservation and Biosafety, Slovak University of Agriculture in Nitra, Tr. A. Hlinku st., 2, Nitra, 94976, Slovak Republic. Tel.: +42-137-641-47-87. E-mail: vladimira.sedlackova@uniag.sk
M. M. Gryshko National Botanical Garden ofUkraine, National Academy of Sciences, Timiryazevska st., 1, Kyiv, 01014, Ukraine. Tel.: +38-067-198-80-82. E-mail: olgrygorieva@gmail.com
Introduction
Horcinova Sedlackova, V., Grygorieva, O., Gurnenko, I., Vergun, O. (2020). Diversity of Sambucus nigra pollen within Slovakia in selected morphological characters by SEM study. Biosystems Diversity, 28(4), 399-404. doi:10.15421/012051
This study analyzed pollen morphology and characteristics of 20 Sambucus nigra L. genotypes from populations of various ecotypes and geographical localities from the territory of Slovakia. We used principal component analysis to explore variability in pollen grain size (polar and equatorial diameter), shape, aperture type, and exine ornamentation by scanning electron microscopy. The combination of these morphological characteristics and ultrastructure allows us to determine the differences or similarities between the same and various species and genotypes, which may be a useful tool for systematics with significant diagnostic value. The findings confirmed small differences among the genotypes in measured traits with polar and equatorial diameters in the range from 22.30 to 26.64 ^m and from 12.81 to 14.45 ^m, respectively. Shape index (P/E ratio) depending on elongation or roundness of pollen grains varied from 1.66 to 2.02. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of morphological data helped to compare evaluated morphometric parameters and identified three closely related groups. It was noted that the diversity of surface sculpturing of pollen grains in combination with their shape and size enables us to use a complex of fine morphologic signs for S. nigra pollen identification. Pollen data combined with other morphological evidence (e.g., floral characters) have more recently become an important indicator of which genotypes may be the best representatives of species.
Keywords: pollen grains; scanning electron microscopy; morphology characteristic; pollen structure.
Carl Nilsson Linnœus first used the term pollen. In the 18th and the early 19th centuries, there was considerable progress in pollen research and the understanding of pollination. The characteristics of pollen, as a useful tool in better understanding of taxonomy and classification of plants, have been studied in terms of the pollen origin (Kozâkovâ & Po-korny 2007), pollen morphology and anatomy (Grew, 1682; Erdtman, 1945; Halbritter et al., 2018), physiology of pollen grain surface (Heslop-Harrison, 1975; Taia et al., 2013), plant sexuality (Camerarius, 1694; Kessler & Harley, 2004), DNA identification (Doyle, 2005; Ziarovskâ et al., 2015), flower pollination (Candolle & Sprengel, 1821; Aluri & Reddi, 1995; Wilcock & Neiland, 2002), pollen germination (Rao & Ong, 1972; Taia et al., 2013), pollen fertilization (Kessler & Harley, 2004; Dressel-haus et al., 2016), characteristics of the offspring (Kolreuter, 1761-1766; Stuessy & Funk, 2013; Ulrich et al., 2012, 2013; Miter et al., 2016), description and figuration of fossil pollen grains and spores (Gee, 2001), medicinal properties (Barbosa et al., 2006; Almaraz-Abarca et al., 2007; Basuny et al., 2013; Nikolaieva et al., 2019).
Pollen grains represent the highly reduced male gametophyte - the enclosing sporoderm and the cellular content, consisting of two or three cells, and the pollen tube (Charzynska & Lewandowska, 1989). The transfer of male gametic material by insects classified the genus Sambucus as entomophilous (Farré-Armengol et al., 2015). It is associated with pollen production, which involves incomparably lower investments in male flowers, but they generally have higher investments in the production of rewards and signals for attracting pollinators (Candolle & Sprengel, 1821; Friedman & Barrett, 2009). Thus, pollen grains adapted to different strategies also have anatomical-morphological differences. These male haploid organisms usually have as variable parameters: the pollen shape and size, the number, type, and position of apertures, and the pollen wall with its
extremely diverse structure and sculpture. The characters of these parameters in comparative pollen (and spore) morphology are at least as important as any other morphological character of the diploid generation (Halbritter et al., 2018). Furthermore, pollen morphological studies proved to be indispensable for the understanding of evolutionary processes and systematics. The ability to identify plants from their pollen has enabled botanists and ecologists to reconstruct past assemblages of plants and identify periods of environmental change (e.g., Fœgri & Iversen, 1989; Moore et al., 1991). Morphological characteristics of pollen grains also can be useful characters in studies of plant taxonomy because many pollen traits are influenced by the strong selective forces involved in various reproductive processes, including pollination, dispersal, and germination (Erdtman, 1952; Nowicke & Skvarla, 1979; Stuessy, 1990; Moore et al., 1991). Thus, the use of pollen morphology as a taxonomic character is challenging, and pollen characteristics must be considered in concert with other characteristics in evolutionary reconstructions.
The genus Sambucus L., with its complex phylogenetic relationships and clades, at first was assigned to the Caprifoliaceae family. In the phylogenetic context it has been occasionally segregated into Sambucaceae, and after cladistic analysis aimed at determining the phylogenetic relationships within Viburnum-Sambucus-Adoxa was classified to the Adoxaceae family by Bremer et al. (2001) classification. Recently, the Nomenclature Committee for Vascular Plants (NCVP) corrected the name for Adoxaceae sensu APG (Applequist, 2013). Comparative pollen revisions of the tribes of the family Caprifoliaceae and their genera have been examined for at least 60 years. The first knowledge was obtained by using an optical light microscope (LM) as evidenced by Erdtman (1952), Straka (1952), Weberling (1966), Stachurska et al. (1970), Kuprianova & Alyoshina (1972). On the base of the modern scanning electron microscope (SEM) many authors have studied pollen sculpture of the Caprifoliaceae family (Accorsi et al., 1987; Hu & He, 1988; Tamas et al., 2009; Tank & Do-
noghue, 2010; Tsymbalyuk & Bezusko, 2017). The study aimed to obtain SEM images of the Sambucus nigra pollen grains in the condition of Slovakia, determine the main morphological traits, and describe the structure of pollen grains in a comparison with literature data. The knowledge of pollen morphological characteristics can be an adequate method for identification of genotypes and allow determination of the differences or similarities between the species.
Materials and methods
Pollen samples and localization. All pollen samples of S. nigra genotypes (SN-01-SN-20) from populations were collected from several parts of Slovakia (Western, South-Western, and Central, Fig. 1). The trees were localized by GPS (altitude of 109-624 m). The whole territory of Slovakia belongs to the temperate climate zone. As the altitude rises, temperature decreases, precipitation increases, soil type change, and the growing season is shorter. The results of meteorological measurements and observations show that the year of sampling (2008) was extremely rich in rain and storm activity throughout the territory, which began to occur in early March. Fresh flowers (not opened) were collected randomly from the different genotypes at the balloon stage (June 2008). Pollen samples released from dry flowers were further dried under laboratory conditions. The dry pollen was used for a microscopic study of morphological characteristics. The samples of pollen grains were applied to double-tape, fastened to metal object tables with a 10 mm diameter.
Fig. 1. Localization of Sambucus nigra L. genotypes within Slovakia using GPS system: the data used to produce this figure can be found in Table I
Morphometric analysis. The measurement of morphometric parameters was carried out on 70 pollen grains from each genotype using the AxioVision Rel. 4.8.2.0 program. The measurements were made in micrometers (pm). The characterization of pollen grains was calculated by taking the following parameters: the polar axis (P - the line connecting the proximal and distal pole), the equatorial axis (E - the line perpendicular to the polar axis and located in the equatorial plane), proximal/equatorial ratio (P/E). The pollen grains were studied at the laboratory of the Department of Tropical and Subtropical plants of NBG using an electron microscope Carl Zeiss LS 15, and the microphotographs were taken. The description terminology has been established with regards to Erdtman (1952), Punt et al. (1974, 2007), Bohnke-Gutlein & Weberling (1981), Halbritter (1998), Halbritter et al. (2018).
The comparative morphological study of the pollen grains was performed according to the working rules on the SEM JEOL JSM-6390 in the conditions of low vacuum (P = 60 Pa) with the following zooming: 500 times - during the measurements; 1,000-10,000 times - while taking the pictures of the exine sculpture features. Using the regime of low vacuum allows the pollen to be studied without its preliminary chemical treatment and undistorted data to received about the research object, which makes the process of the probe preparation easier. Typical exine patterns, shape, size, and the dimensions of pollen grains for each S. nigra genotype were determined by using a scanning electron micrograph (SEM).
Statistical analyses. Basic statistical analyses - the minimal and maximal values of the traits, arithmetic means, and coefficient of variation (CV, %) were performed using PAST 2.17. Results of the morphometric analy-
sis were determined by mean ± standard deviation (SD) and statistical significance was estimated. The level of variability was determined by Stehlikova (1998). Pearson's correlation coefficient was used to depict the relationship between the two traits. Hierarchical cluster analyses of similarity between phenotypes were computed by the Bray-Curtis similarity index and were performed using PAST 2.17. Principal component analysis (PCA) was performed to evaluate relationships among variables and some possible genotype groupings based on similar properties by using XLSTAT procedure (XLSTAT 7.5, Addinsoft, USA). All the observed traits were shown in graphic form.
Table 1
Localities of Sambucus nigra L. genotypes in Slovakia and their altitude
Genotype Locality Region of Slovakia Altitude, m a.s.l.
SN-01 Mosovce Central Slovakia 484
SN-02 Blatnica Central Slovakia 495
SN-03 Topol'niky South-Western Slovakia 111
SN-04 Blatnica Central Slovakia 495
SN-05 Budmerice-Ruzindol Western Slovakia 176
SN-06 Trnava Western Slovakia 144
SN-07 Cabaj-Capor Western Slovakia 159
SN-08 Horna Stubna Central Slovakia 624
SN-09 Ziharec South-Western Slovakia 111
SN-10 Bartosova Lehôtka Central Slovakia 390
SN-11 Sturovo South-Western Slovakia 109
SN-12 Topol'niky South-Western Slovakia 111
SN-13 Kamenica nad Hronom South-Western Slovakia 132
SN-14 Vrable Wester Slovakia 144
SN-15 Chl'aba South-western Slovakia 115
SN-16 Topolniky South-western Slovakia 111
SN-17 Sturovo South-western Slovakia 109
SN-18 Pohronsky Ruskov South-western Slovakia 131
SN-19 Malé Zaluzie Western Slovakia 160
SN-20 Malé Zaluzie Western Slovakia 160
Note, altitude - meters above sea level.
Results
Pollen shape refers to the 3-dimensional form of a pollen grain concerning the P/E ratio. Results of 20 tested genotypes of S. nigra pollen morphology were calculated for the polar (p.a.) and equatorial axis (e.a.), which showed that dry pollen grains are generally from small (10-25 pm) to medium size type (25-50 pm), in accordance with dimensions and P/E ratio (Fig. 2) pollen grains are frame in prolate type. Their shape was characterized as tricolporate, oblate or spherical, sometimes oblate-spheroidal, in polar view pollen was 3-lobate and in equatorial view elliptical or circular.
An aperture is a region of the pollen wall that differs significantly from its surroundings in morphology and/or anatomy. The aperture is presumed to function as the site of germination and to play a role in harmo-megathy - the mechanism permitting changes in the shape and size of the pollen grain due to varying hydration status. Colpi (elongated apertures) (p.a/e.a ratio > 2) were long, situated equatorially or globally distributed, on the edges more or lesser equal and clear edges with slightly pointed ends. Membranes of colpi were psilate with a smooth surface. Mainly pores were blurred, covered with margins of colpi. But in very rare cases pores were distinct.
In general depending on the pollination the outer pollen wall may be either highly ornamented, often with plenty of pollen coatings, mainly pollenkitt, or with a more or less of psilate pollen surface. The pollen wall of the tested samples was with tectum. Ectexine consisted of obvious, short, and thin rod-shaped reinforcing elements. They were sparsely located. The exine surface had verrucate and baculate sculpting with rounded cells. Sculpture of exine was reticulate or microreticulate or all over pollen surface or sometimes with foveolate apocolpium. Luminae were irregularly shaped, rather small in polygonal (often 4- or 5-angular) or rounded, rather small in mesocolpium; luminae decreasing in size towards the colpi and the apocolpium. Cells were small or medium size, circular, angled, or circular-angled by the shape. Sometimes at the bottom of cell columns are observed.
Fig. 2. Pollen grains of Sambucus nigra L. species in different positions A-B. Groups ofpollen grains; Scale bar = 10 pm x 400 and 10 pm x 800.
C-D. Dry pollen grains in reticulate apocolpia; Scale bar = 1 pm x 5000. E-F. Detail of exine surface microreticulate ornamentation ofmesocolpium, within lumina free columellae visible; Scale bar = 1 pm x 10 000
Pollen measurements for each genotype are listed in Table 2. The total length of the polar axis (P) varied in the range of 18.23-29.91 pm, the total width of the equatorial axis (E) was in the interval 10.86-16.83 pm (Table 2). The values of the variation coefficient show the values 2.628.62% for polar axes and 4.40-7.17% for equatorial axes. Results of the morphometric analysis were also determined by mean ± standard deviation (SD) and statistical significance was estimated. Figure 3 shows mean values of polar diameters in the range from 22.30 ± 1.92 pm (SN-05) to 26.64 ± 0.70 pm (SN-01) and equatorial diameters from 12.81 ± 0.74 pm (SN-06, SN-17) to 14.45 ± 0.81 pm (SN-08), respectively. Means in each column are not significantly different (P < 0.05). The shape index (P/E ratio) depends on the elongation or roundness of pollen grains. Our samples' mean P/E ratio varied from 1.66 ± 0.18 (SN-05) to 2.02 ± 0.11 (SN-10) (Fig. 4). Measurements showed that average values for polar axes are larger than 25 pm (small-medium sized) for 6 genotypes (SN-01, SN-08, SN-10, SN-11, SN-14, and SN-18) in various localities.
S. nigra grains have prolate spheroidal, subprolate, or slightly oblate (oblate spheroidal) shape (Maciejewska, 1997). The sizes of pollen grains S. nigra are very similar, the same for P/E ratio from 1.65 ± 0.18 (SN-05) to 2.02 ± 0.11 (SN-10), whereas lengths of p.a. and e.a. are in major intervals. The data indicates that hierarchical cluster analysis (HCA) separates pollen selections into three closely related groups - main clusters. Cluster III contains most genotypes that have nearly identical parameters. Other authors have also used cluster analysis to evaluate the morphological data of pollen, including pollen characteristics facilitating a better understanding of taxonomic classification, the genus or subgenus relationships or phylogenetic lineages (Grygorieva et al., 2010; Oswald et al., 2011; Teleb & Salah-El-Din, 2014; Baldemir et al., 2018; Soares et al., 2018).
Table 2
Variation limits ofpollen morphological traits of selected genotypes of Sambucus nigra L.
P - Polar E - Equatorial SI - shape
Genotypes axis, pm axis, pm index (P/E)
min max V, % min max CV, % min max V, %
SN-01 25.61 28.68 2.60 12.61 15.64 4.61 2.03 1.84 1.77
SN-02 22.07 27.52 4.08 12.31 15.88 4.96 1.79 1.73 1.22
SN-03 20.76 25.17 4.74 11.92 15.92 6.25 1.74 1.58 1.32
SN-04 22.56 26.70 3.35 11.84 15.01 5.07 1.91 1.78 1.51
SN-05 18.23 26.88 8.56 10.86 16.83 7.12 1.68 1.60 1.20
SN-06 18.75 25.95 5.80 11.06 15.71 6.54 1.70 1.65 1.13
SN-07 19.04 27.22 7.13 11.68 15.46 6.08 1.63 1.76 1.17
SN-08 23.19 29.91 4.28 12.80 16.62 5.56 1.81 1.80 1.30
SN-09 22.66 25.92 3.11 12.09 15.77 5.16 1.87 1.64 1.66
SN-10 23.42 28.32 2.96 11.81 15.72 4.70 1.98 1.80 1.59
SN-11 23.72 26.93 2.64 10.94 14.90 5,14 2.17 1.81 1.95
SN-12 21.42 25.80 4.23 11.78 14.89 4.37 1.82 1.73 1.03
SN-13 22.68 26.64 3.60 11.40 14.77 4.72 1.99 1.80 1.31
SN-14 23.85 27.06 2.97 11.16 14.43 4.68 2.14 1.88 1.58
SN-15 21.72 26.48 3.91 11.81 15.61 5.65 1.84 1.70 1.45
SN-16 22.38 26.51 2.96 11.74 15.26 4.57 1.91 1.74 1.54
SN-17 21.43 26.20 3.68 11.15 15.18 5.73 1.92 1.73 1.56
SN-18 22.48 28.55 4.12 12.18 15.86 5.72 1.85 1.80 1.39
SN-19 20.99 26.80 4.66 12.28 15.91 5.61 1.71 1.68 1.20
SN-20 21.43 26.85 4.60 12.31 16.43 5.66 1.74 1.63 1.23
Note, min - minimal value; max - maximal value; V - coefficient of variation (%).
30 2? 20 Ii 10
ab , b
bc
ab
^ s^ <ste 5? s? O O s^ O -i
# # # # # # # #'
Genotypes
H Polar axis, inn Li Equatorial axis, inn
Fig. 3. Diameter values ofpolar axis (P) and equatorial axis (E) of Sambucus nigra L. pollen samples: means in each column followed by different letters are not significantly different (P < 0.05)
ab , I b
bc bc
bc bc bc be t
ab
bc
b ab ab ab ab
t b , T „ bc
T
^ <iv <S> sf> ç?' s? s> vfe _<>
G«i(ilj|>es
Fig. 4. Diameter values of shape index (P/E) of Sambucus nigra L. pollen samples: means in each column followed by different letters aie not significantly dilferent (P < 0.05)
The results of the cluster analysis simply illustrate the groupings of genotypes with similar morphological characters. Based on the cluster analysis we made a dendrogram of polar axes, equatorial axes, and shape index (Fig. 5).
Fig. 5. Cluster dendrogram of morphometric parameters pollen of Sambucus nigra L. genotypes
More detailed relationships between genotypes were revealed by principal component analysis (PCA). The PCA used in our work showed that 100.0%o of the variability observed was explained by the first two components (Table 3). PC1, PC2, and PC3 accounted for 61.7%, 3.3% and 0.01% respectively. PC1 was positively correlated with the polar axis and shape index. PC2 was positively correlated with the polar and equatorial axis, whereas the shape index showed a very low negative correlation. Positive values for PC1 correspond to the genotypes with a higher polar axis as shown in Figure 5. Genotypes SN-01, SN-10, and SN-18 were included in this group. The highest negative values for PC1 indicate the genotypes with the smallest polar axis. This group includes genotypes SN-05 and SN-06 (Fig. 6). The genotypes SN-08 and SN-19 which the highest PC2 due to the highest equatorial axis. The positive PC3 value indicates the largest shape index. These characteristics were observed in genotype SN-10.
Table 3
Eigenvalues and proportion of total variability, eigenvectors of the three principal components (PC), and component scores for Sambucus nigra L. pollen 20 genotypes
Selection Component scores
PC1 PC2 PC3
Eigenvalue 1.852 1.148 0.000
Variance (%) 61.730 38.257 0.013
Cumulative 61.730 99.987 100.000
Variable Component loadings
PC1, X=61.73 PC2, X=38.25 PC3, X=0.01
Polar axis, ^m 0.577 0.578 -0.577
Equatorial axis, ^m -0.360 0.814 0.456
SI - shape index 0.733 -0.056 0.677
Discussion
Punt et al. (1974, 1976) divided the family Caprifoliaceae into 10 distinct pollen types viz., and determined that Sambucus nigra-type pollen grains aie (2-), 3-Zonocolporate, P/E ratio is suberect to erect; ectoaperture -colpus, long and wide, deeply sunken; end acute to slightly obtuse; bridge either distinct or indistinct and narrow; membrane nudate; endoaperture -lalongate colpus, rather indistinct without costae; ornamentation - reticulate; lumina irregularly shaped, rather small in mesocolpium; lumina decreasing in size towards the colpi and the apocolpium; muri simplico-lumellate; columellae in surface view circular; equatorial view - circular to elliptic; Polar view - circular or triangular with convex sides and apertures situated in the obtuse angles.
Maciejewska (1997) described pollen type according to P/E ratio as prolate polar axis predominate (prolate spheroidal - 30.5% of observed pollen grains, subprolate - 44.8%, prolate - 9.6%); slightly oblate ones constitute no more than 1/5 of observed samples (oblate spheroidal -18.1%). Hexagonal polar view, with convex sides, elliptic equatorial view, sometimes also circular. Reticulate all over pollen surface or sometimes with foveolate apocolpium, muri simplicolumellate with smooth ridges.
Biplot (aits PCI aii<lPC2: 99.98 »/o)
SN-08 • SN-19 • SN-20 • • SN-02 SN-09 *. . SN-0" • SN-01 • SN-1S ' SN-15
SN-03 • . SN-12 5 • SN-06 • . .SN.„ 'SN SN-13 • SN-14 • SN-16 • SN-17
-3 -2-10123 axis PCI (61.73%)
Fig. 6. Biplot based on principal components analysis (PCA) for pollen morphometric parameters of Sambucus nigra L. 20 genotypes
Table 4
Literature data on pollen morphometric parameters in the Sambucus nigra L.
Characteristic Value Autors Country
19.0-26.0 Punt et al., 1974 Netherlands
16.0-24.8 Maciejewska, 1997 Poland
25.0 Muccifora et al., 2003 Italy
24.25 Tamas et al., 2009 Romania
Polar 15.9-21.3 Tsymbalyuk and Bezusko, 2017 Ukraine
axis, pm 22.11-27.07 Horcinova Sedlackova et al., 2018 Ukraine
14.00-28.00 Wronska-Pilarek et al., 2020 Poland
18.00-22.00 Wronska-Pilarek et al., 2020 Germany, Lithuania
18.23-29.91 Our data Slovakia
15.0-22.0 Punt et al., 1974 Netherlands
16.0-22.0 Maciejewska, 1997 Poland
12.5 Muccifora et al., 2003 Italy
12.85 Tamas et al., 2009 Romania
Equatorial 13.3-18.6 Tsymbalyuk and Bezusko, 2017 Ukraine
axis, pm 11.98-17.29 Horcinova Sedlackova et al., 2018 Ukraine
12.00-24.00 Wronska-Pilarek et al., 2020 Poland
16.00-22.00 Wronska-Pilarek et al., 2020 Germany, Lithuania
10.86-16.83 Our data Slovakia
1.02-1.50 Punt et al., 1974 Netherlands
0.89-1.42 Maciejewska, 1997 Poland
1.88-1.89 Tamas et al., 2009 Romania
SI - shape 1.70-1.76 Horcinová Sedlácková et al., 2018 Ukraine
index 0.82-1.57 Wroñska-Pilarek et al., 2020 Poland
0.90-1.25 Wroñska-Pilarek et al., 2020 Germany, Lithuania
1.08-2.36 Our data Slovakia
Wronska-Pilarek et al. (2020) presented pollen sculpture as reticulate-heterobrochate, mostly micioieticulate-heterobiochate pollen wall with lumina of different, often irregular shape; within the lumina one to three free columellae, shorter than muri, often present. Outside the colpori, the exine ornamentation was microreticulate or microgranulate and in the middle of the colporus, it was usually psilate.
Almost in all literature data (Table 4), S. nigra pollen grains were described as small (10-25 pm). Tämas et al (2009), who examined in the Romanian flora and vegetation three species of Sambucus (S. nigra, S. racemosa, S. ebulus) by morphological traits by SEM analysis, determined that only S. ebulus had over 25 pm as middle size type (2550 pm), Maciejewska (1997) evaluated the Polish species of Sambucus (S. nigra, S. racemosa, S. ebulus) with low polar diameter for all samples. Wronska-Pilarek et al. (2020) identified a total of 66 evaluated samples in Poland, Germany, and Lithuania, almost all the pollen grains were small, with just one medium-sized. S. nigra grains have prolate spheroidal, sub-prolate, or slightly oblate (oblate spheroidal) shape (e.g. Donoghue, 1985; Maciejewska, 1997; PalDat, 2020). The sizes of pollen grains S. nigra are very similar, whereas lengths of p.a. and e.a. are in major intervals, as well
as measurements of Punt et al. (1974), Horcinová Sedlácková et al. (2018) or Wroñska-Pilarek et al. (2020). Simultaneously & Maciejewska (1997), Tsymbalyuk & Bezusko (2017), Wroñska-Pilarek et al. (2020) presented small differences between polar and equatorial intervals, which is related to the lesser values of SI-shape index (p.a./e.a. ratio). Studies of pollen samples from Slovakia and Ukraine (Horcinová Sedlácková et al., 2018) have presented wide ranges of pollen diameters, P/E ratio (1.08-2.36) of Slovakian samples against the authors' study of Ukrainian samples (p.a./e.a. = 1.70-1.76) and other data ofPunt (1974), Maciejewska (1997), Muccifora et al. (2003), Tamas et al. (2009) or Wroñska-Pilarek et al. (2020) (Table 3).
Pollen size is an important diagnostic feature in the identification and characterization of hybrid offspring. Differences in pollen grain size may also occur between different genotypes of the same species (Miter et al., 2016). The size of the pollen grains can also vaiy within a single plant, the largest pollen grains are found in the best developed top inflorescences. The data of the authors on the size of the pollen grains of the same plant species are variable and may vary, often depending on the pollen treatment in the analyses. Various external factors (air temperature, precipitation) and plant nutrition can influence the size of pollen grains. Plants are known to produce fewer pollen grains but larger sizes to avoid desiccation stress during the flowering period (Ejsmond et al., 2011).
Palynological features are very valuable. Evaluated pollen characters may be a useful tool for systematics with significant diagnostic value. Pollen data combined with other morphological evidence (e.g., floral characters) have more recently become an important indicator of which genotypes may be the best representatives of species (Ulrich et al., 2012, 2013; Stuessy & Funk, 2013).
Conclusions
The study of the S. nigra pollen via scanning electron microscope allowed us to determine the most important parameters which can be used to identify the representatives of species. The detailed pollen morphological and micro-sculptural characteristics were investigated, described, and analyzed by using a hierarchical cluster analysis dendrogram and BiPlot. The main parameters such as the form (the pollen grains' elongation, P/E ratio) are specific for different Sambucus species. Results from our analyses showed small differences among S. nigra genotypes. Some of these pollen morphological parameters can be used for identification and comparison with the following analyses of Sambucus species.
The publication was prepared with the active participation of researchers involved in the international network AgroBioNet of the institutions and researchers for the realization of research, education, and development program "Agrobiodiversity for improving nutrition, health, and life quality". This work was supported by the Bilateral Scholarship of the Ministry of Education, Science, Research and Sport (Slovak Republic) and Visegrad Fund.
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