The optimal sample size in pollen morphological studies using the example of Rosa canina L. (Rosaceae)

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1 Palynology, 2015 Vol. 39, No. 1, 56 75, The optimal sample size in pollen morphological studies using the example of Rosa canina L. (Rosaceae) Dorota Wronska-Pilarek a *, Andrzej M. Jagodzinski b, Jan Bocianowski c and Magdalena Janyszek d a Department of Forest Botany, Poznan University of Life Sciences, Wojska Polskiego 71d, Poznan, Poland; b Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, Kornik, Poland; Department of Forest Protection, Poznan University of Life Sciences, Wojska Polskiego 71c, Poznan, Poland; c Department Mathematical and Statistical Methods, Poznan University of Life Sciences, Wojska Polskiego 28, Poznan, Poland; d Department of Botany, Poznan University of Life Sciences, Wojska Polskiego 71c, Poznan, Poland This study undertook an investigation of an important problem, so far completely overlooked in the palynological literature to determine the optimal sample size for pollen grain morphological studies. In other words, we investigated the number of pollen grains which should be measured in order to obtain a representative mean value of a given quantitative feature which, in consequence, would make it possible to more accurately describe the pollen of a given taxon. Investigations were conducted on a sample comprising 3850 Rosa canina L. pollen grains on the basis of the length of the polar axis (P), the equatorial diameter (E) and the P/E ratio, at the flower, specimen and population levels. The size of the pollen samples analysed reflected common sample numbers employed in previous pollen morphology studies, namely from five through 10, 20, 30, 50, 100 up to 200 pollen grains. The statistical analyses performed revealed a relatively low variability in pollen grain biometric features at the levels of flower, specimen and population. At the lowest level of variability analysed, it is sufficient to take measurements of several grains to obtain values satisfactorily representing the variability within the flower level. At the level of a specimen or population, the number of grains necessary to secure representative mean values should range from 15 to 20. However, when the research objective is not only information regarding mean values of pollen grain biometric features but also the analysis of their variability (min max), then the sample size should include approximately 30 grains. The results obtained, apart from their significance in taxonomic studies, also possess important practical significance; measurements of pollen grain biometric features are very labour-intensive and costly and, sometimes, because of difficulties in obtaining satisfactory quantities of plant material (e.g. herbarium specimens, rare species, paleopalynological collections), also very sparse. Keywords: pollen sample size; pollen morphology; intraspecific variability; Rosa; statistical analysis 1. Introduction The palynological literature appears to overlook a very important problem of sample size, i.e. the number of pollen grains which ought to be measured in order to secure a precise description of pollen grain features of a given taxon. There are no methodological guidelines regarding the optimal pollen sample size for morphological investigations. This probably explains why numbers of pollen grains in samples in individual research papers vary greatly; sometimes the numbers are imprecise or omitted altogether. For instance, in most recent papers describing pollen morphology of taxa derived from many different genera and families, sizes of pollen samples range from 10 (Isik & D onmez 2006; Kevin et al. 2008) through about (Alwadie 2008; Juan & Nian-He 2011), 20 (Borges et al. 2009; Jia-Xi et al. 2010), 25 (Lima et al. 2010), 30 (Remizowa et al. 2008; Wronska-Pilarek et al. 2010; Ozler et al. 2011), 50 (Isik & D onmez 2006; Rodondi et al. 2010), up to 400 (Pipino et al. 2011) and pollen grains (Naruhashi & Takano 1980). There are also publications in which no numbers of measured pollen grains are provided (e.g. Herber 2002; Polevova et al. 2010; Punekar & Kumaran 2010). Investigations were carried out on Rosa canina L. because it is one of the most common species from the Rosa genus, characterised by a wide natural range of occurrence, investigated thoroughly and considered a good species, both in classical taxonomy (Zielinski 1985, 1987; Henker 2000; Kalkman 2004) as well as from the point of view of pollen morphology (Reitsma 1966; Eide 1981; Hebda & Chinnappa 1994; Wronska- Pilarek 2011; Wronska-Pilarek & Jagodzinski 2011). Based on our previous studies (Wronska-Pilarek & Jagodzinski 2011, 2012; Wronska-Pilarek 2011) the general characteristics of R. canina pollen morphology are well described. Pollen grains of R. canina are monad and isopolar. The pollen size is medium: mean * Corresponding author. pilarekd@up.poznan.pl Ó 2014 AASP The Palynological Society

2 Palynology 57 length of polar axis (P) equals (range: ) mm, equatorial diameter (E) is ( ) mm and the P/E ratio is 1.24 ( ). The pollen shape is mostly subprolate, less frequently prolate and prolate-spheroidal. Outlines in equatorial view are mostly elliptic and, in polar view, circular. The exine has a mean thickness of 1.61 m. Exine sculpturing is striate. Striae usually run parallel to the polar axis but frequently they also form loops. At the bottom of the grooves, circular or elliptic perforations are found. The pollen grains of R. canina are colporate with three (very rarely four) ectocolpi and with the same number of endopores, usually located in the middle of the ectocolpi. The ectocolpi are arranged meridionally, regularly. They are acute, elliptic in outline and short (on average: 20.5 m). The R. canina ectocolpus is crossed by a bridge at the equator, dividing it into two parts, formed by two intersecting bulges of ectexine. Above the middle part of the ectocolpus an operculum is also present a convex, large and wide structure. As an example, the pollen grains of Rosa canina are shown in a few scanning electron microscope (SEM) photographs (Plate 1, figures 1 3). There is no doubt that the selection of only one species for investigation means that the results obtained will not be universally relevant. Nevertheless, we assume that the results regarding sample size will be applicable at least for species with colporate pollen, similar in structure to R. canina from the colporate pollen class. The choice of two pollen features for analyses, namely the length of the polar axis (P) and the equatorial diameter (E), is by no means accidental. Earlier studies on many Rosa species have shown that they are widely considered to be the least variable quantitative pollen grain features at both individual and population levels (Wronska-Pilarek & Jagodzinski 2009, 2011). This is why these two features are most frequently analysed in palynological studies concerned not only with pollen morphology and variability of different taxa (e.g. Dıaz Lifante 1996; Torres 2000; Alwadie 2008; Meltsov et al. 2008; Cruden 2009; Wronska-Pilarek & Jagodzinski 2009, 2011) but also those dealing with pollen viability (e.g. Kelly et al. 2002; Gupta et al. 2008; Kormutak et al. 2008; Marciniuk et al. 2010), genome size (e.g. Knight et al. 2010), cytology (e.g. Singhal & Kumar 2008) or ploidy level (e.g. Jacob & Pierret 2000; Pradeep & Jambhale 2000). The goal of this investigation was to determine the optimal sample size required for pollen morphological studies, i.e. the number of pollen grains which need to be measured in order to obtain a representative mean value of a given quantitative feature and which, consequently, would make it possible to appropriately describe the pollen of a given species. Experiments were carried out on a large sample (3850 pollen grains) on the basis of statistical analyses of the following three quantitative pollen grain features of Rosa canina: length of the polar axis (P), equatorial diameter (E) and P/E ratio, at the flower, specimen and population levels. Our intention was to draw the attention of palynologists to this important methodological problem as well as to initiate a discussion of this subject. We sincerely hope that our investigation will encourage researchers to standardise the size of pollen samples used in palynological studies. 2. Methods Flowers of Rosa canina were collected from 18 shrubs growing in the outskirts of Poznan, along a distance of about 3 km on Kobylepole and Sowice streets ( N, E). Shrubs were growing on both sides of the streets at distances of several to several dozen meters. Four flowers were collected randomly from each of the 18 R. canina shrubs (72 flowers in total). Only flowers which had just bloomed or were about to bloom were used in the study. A sample consisted of 50 correctly formed pollen grains derived from a single flower (200 pollen grains in total per each individual). To precisely estimate the variability of pollen morphological features inside the flower, we collected 100 pollen grains from five randomly selected flowers harvested from five different specimens. The maximal sample sizes in our study (50 or 100 pollen grains per flower) were used because they matched maximal sample sizes from previously published papers by other authors. In total, 3850 pollen grains were studied. The size of pollen samples reflected all variants employed by palynologists, namely from five through 10, 20, 30, 50, 100 and up to 200 pollen grains. All samples were acetolysed according to Erdtman s method (1960). The acetolysing mixture was made up of nine parts of acetic acid anhydride and one part of concentrated sulphuric acid and the process of acetolysis lasted 2.5 minutes. The pollen samples were prepared for light microscopy (using a Biolar 2308 light microscope) in glycerine jelly. Pollen grains were measured using the light microscope (640 ) and the eyepiece (ocular) with a scale and then measurement results were recalculated into micrometres by multiplying them by 2. SEM was used to take photographs and to describe qualitative pollen features. For SEM investigations, pollen were mounted in 96% ethyl alcohol. All samples were mounted on stubs with double-faced adhesive tape, sputter coated with gold, and examined using a Hitachi S-3000N scanning microscope.

3 58 D. Wronska-Pilarek et al. Plate Scanning electron microphotographs of R. canina pollen grains: 1. equatorial view with two colpi end bridges; 2. polar view with tree colpi; 3. striate exine sculpture; striae and grooves with the perforations visible. Pollen grains were analysed for three quantitative features, i.e. length of polar axis (P), equatorial diameter (E) and P/E ratio. The palynological terminology of Punt et al. (2007) and Hesse et al. (2009) was used in this paper. To estimate the number of pollen grains needed for precise estimation of mean values of the features P, E and P/E, we took five different shrubs, one flower from every shrub and 100 pollen grains from each flower (500 pollen grains in total; shrub numbers as follows: one, six, 11, 15 and 18). Based on 100 pollen grains for each flower, we calculated mean values of the pollen grains features and 5% range from the mean. We calculated cumulative mean value of each pollen grain feature according to the formula: CM i ¼ðx 1 þ...þ x i Þ=i (1) where CM is cumulative mean value, and x 1 to x i are values obtained for the 1 st to i th pollen grain. The order of pollen grains for each flower used for the analysis was random. For each pollen grain feature, one-factor analysis of variance (ANOVA) was used to examine differences in the mean values obtained based on five, 10, 20, 30, 50 and 100 pollen grains collected from each flower (five) for each Rosa canina individual separately. When critical differences were noted, multiple comparisons

4 Palynology 59 Figure 1. Cumulative mean values of length of polar axis (P, mm) of pollen grains for 5 individuals of R. canina. P values were estimated based on 100 pollen grains collected from one flower from each of 5 individuals. The order of pollen grains used in the analysis was random. were carried out using Tukey s test for equal sample sizes. Moreover, to examine the variability in the mean values of pollen grain morphological features obtained at the individual and population level, we took one, two, three, four, five, seven, 10, 20, 30, 40 and 50 pollen grains from each flower (four) for each shrub individually (18 shrubs). Thus we estimated mean values of the pollen features based on four, eight, 12, 16, 20, 28, 40, 80, 120, 160 and 200 pollen grains for each specimen and mean values of the pollen features based on 72, 144, 216, 288, 360, 504, 720, 1440, 2160, 2880 and 3600 pollen grains at the population level. We compared mean values of each pollen grain feature at the specimen (200 pollen grains) and population (3600 pollen grains) levels with values obtained based on variable sample sizes of pollen grains used for the analysis

5 60 D. Wronska-Pilarek et al. Figure 2. Cumulative mean values of length of equatorial diameter (E, mm) of pollen grains for 5 individuals of R. canina. Analysis description same as in Figure 1. (max:mean and min:mean expressed in %) to show maximal and minimal differences between these values. Additionally, one-way ANOVA was used to examine differences in the mean values obtained based on variable sample sizes at the specimen and population levels. Moreover, we calculated mean values and coefficients of variation (Kozak et al. 2013) for each pollen grain feature studied when five, 10, 20, 30, and 50 randomly selected pollen grains were used (from the database of 3600 pollen grains) and compared the values with the respective means calculated based on all pollen grains collected (3600). In total, 10 drawings were done. To determine the pollen size which is reliable for morphological analysis at the population level, the values which were in the range 5% of the respective population means were noted. These statistical analyses were performed using JMP 10.0 (SAS Institute Inc. Cary, NC. USA;

6 Palynology 61 Figure 3. Figure 1. Cumulative mean values of P/E ratio of pollen grains for 5 individuals of R. canina. Analysis description same as in

7 62 D. Wronska-Pilarek et al. Thirty subsets comprising five, 10, 20, 50 and 100 pollen grains were randomly selected for each shrub. Treating the sample size as a potential differentiating factor, ANOVA was performed to verify the general zero hypothesis concerning the lack of sample size impact on values of the observed feature. The analysis was carried out for P, E and P/E features. Next, on the basis of 30 observations, expected value estimators were assessed for individual sample sizes of five, 10, 20, 50 and 100 pollen grains, and the mean value for the entire population comprising 200 pollen grains was calculated. Values of the least significant difference (LSD 0.05 ) were estimated and, on their basis, detailed testing was performed to demonstrate which sizes differed (or did not differ) significantly from the size of 200. The enclosed boxplots present minimal and maximal values and the median as well as bottom and top quartiles of the features analysed, taking into account sample sizes. These data analyses were performed using the statistical package GenStat Results Based on cumulative mean values of P, E and P/E ratio established on the basis of 100 pollen grains collected from one flower from each of five individuals of R. canina (shrubs no. 1, 6, 11, 15 and 18), it was shown that mean values ( 5%) at the flower level are generally obtained when a few to several pollen grains were analysed (Figures 1 3). The minimum number of pollen grains for reliable estimation of mean values of P, E and P/E ratio is ca. 10 pollen grains. When other drawings of pollen grain order were made to analyse cumulative mean values of particular pollen grain morphological features, the results were similar to those shown in Figures 1 3. When estimating mean values of pollen morphological features studied based on five randomly selected flowers collected from five different individuals of R. canina shrubs, it was shown that there were no statistically significant differences between means calculated based on different pollen sample sizes (i.e. five, 10, 20, 30, 50 and 100) (Table 1). It is evident that to establish the representative mean value of a given pollen feature for a particular flower, five pollen grains might be used. However, this number of pollen grains might be insufficient for estimation of pollen grain variability among flowers within a specimen; thus, it was decided to analyse 50 pollen grains per flower from four flowers representing each specimen to show the potential differences in pollen grain morphology at the specimen and population levels. Because the recommended sample size in statistical studies is 30, we decided to compare it with samples comprising five, 10 and 20 pollen grains for two basic Table 1. Mean values of pollen morphological features ( standard error, SE) of Rosa canina determined for five individuals based on variable pollen grain sample sizes (100 pollen grains from one flower per specimen). One-way analyses of variance (ANOVAs) were performed separately for each individual shrub to determine the differences among mean values of particular pollen grain morphological features when variable sample sizes were used. Pollen grain feature Shrub no. Sample size P (mm) E (mm) P/E (1.47) (0.40) (0.048) (0.85) (0.52) (0.027) (0.53) (0.33) (0.018) (0.38) (0.32) (0.015) (0.28) (0.29) (0.013) (0.20) (0.22) (0.010) ANOVA P > F F P (1.10) (0.75) (0.043) (0.68) (0.78) (0.035) (0.57) (0.60) (0.031) (0.48) (0.42) (0.023) (0.34) (0.34) (0.018) (0.22) (0.27) (0.016) ANOVA P > F F P (0.98) (0.98) (0.084) (0.67) (0.87) (0.050) (0.49) (0.51) (0.031) (0.43) (0.38) (0.023) (0.29) (0.30) (0.018) (0.23) (0.26) (0.016) (continued)

8 Palynology 63 Table 1. (Continued ) Pollen grain feature Shrub no. Sample size P (mm) E (mm) P/E ANOVA P > F F P (1.02) (0.89) (0.039) (0.78) (0.58) (0.029) (0.50) (0.38) (0.019) (0.39) (0.35) (0.016) (0.31) (0.25) (0.012) (0.22) (0.18) (0.009) ANOVA P > F F P (0.75) (0.80) (0.033) (0.52) (0.45) (0.018) (0.47) (0.39) (0.014) (0.38) (0.29) (0.011) (0.26) (0.21) (0.008) (0.22) (0.18) (0.010) ANOVA P > F F P Notes: P - length of polar axis, E - equatorial diameter. features (P and E) in all 18 individuals (shrubs) (Table 2). In the majority of cases, an absence of statistically significant differences in mean P and E values was found between samples containing five, 10 and 20 pollen grains and the reference sample embracing 30 pollen grains. For example, for the sample comprising five pollen grains, the P feature values were most divergent in comparison with those recorded for the reference sample size; i.e. in eight among 18 individuals, statistically significant differences occurred in comparison with the sample comprising 30 pollen grains. When 10 or 20 pollen grains were used in the analysis, the differences in mean values of P with samples of 30 pollen grains were noticed only for five or four individuals. Table 2. Comparison of the mean values of P and E of pollen samples determined for 5, 10 and 20 pollen grains with mean values determined based on recommended number of observations in statistical analyses (30 pollen grains). Abbreviations: ID, no statistically significant differences (at the level of at least 0.05); SD, shading statistically significant differences. Shrub no. Size of pollen P (mm) E (mm) sample ID ID ID ID ID ID 2 SD SD SD ID SD ID 3 SD SD SD SD SD SD 4 ID ID ID ID ID SD 5 SD ID ID ID ID ID 6 ID ID ID SD SD SD 7 SD SD SD SD SD SD 8 ID ID ID ID ID ID 9 SD SD ID ID ID ID 10 ID ID ID ID ID ID 11 SD SD SD ID ID ID 12 ID ID ID ID ID ID 13 ID ID ID ID ID ID 14 SD ID ID SD ID ID 15 ID ID ID ID ID ID 16 ID ID ID ID ID SD 17 ID ID ID ID ID SD 18 SD ID ID SD SD ID On the boxplots (Figures 4 6), for all of the features examined, the sample embracing five pollen grains differed most from the remaining samples because it was characterised by the greatest variability range (from minimum to maximum). This also refers, although to a lesser extent, to the samples comprising 10 pollen grains. The statistical analyses for samples comprising 20, 50 and 100 pollen grains revealed an absence of statistically significant differences with respect to the population average (for 200 pollen grains). Based on the mean values of all morphological features studied, determined for each specimen separately on four to 200 pollen grains, we analysed percentage differences among mean values determined on the basis of 200 pollen grains, and means calculated based on variable sample sizes (Tables 3 5). In general, there were no statistically significant differences in means of pollen grain morphological features at the specimen level, when variable sample sizes of pollen grains (4 200) were used for the analyses. For example, maximal mean length of P of shrub no. 1 (32.50 mm; one pollen grain per flower, four pollen grains per specimen) was 1.6% higher than the mean value calculated on the basis of 200 pollen grains, whereas minimal

9 64 D. Wronska-Pilarek et al. Figure 4. Boxplot of the relationship between values of length of polar axis (P, mm) and pollen sample size. Boxplot was created by putting the analysed parameter distribution on the x axis. The left side of the box is determined by the lower quantile, Q 1, and the right side of the by the upper quantile, Q 3. The length of the box corresponds to interquartile range. The vertical line inside the box corresponds to median value. The end of the left section determines the minimum value in a set, while the right end the maximum value of the trait. mean length of P of shrub no. 1 (31.67 mm; three pollen grains per flower, 12 pollen grains per specimen) was 1% lower than the mean value calculated on the basis of 200 pollen grains (31.99 mm; Table 3). The highest difference for the P value was found for shrub no. 12 (maximal value was 5.9% higher than the mean value) and for shrub no. 16 (minimal value was 6% lower than the mean value). At the population level, the difference between the minimal value of P (e.g. obtained based on 12 pollen grains per shrub; 216 pollen grains in total) and the mean value obtained based on all 3600 pollen grains was 0.99%. For 16 out of 18 shrubs studied, the differences in mean P values obtained based on four, eight, 12, 16, 20, 28, 40, 80, 120 and 160 pollen grains were lower than 5% of the mean value obtained based on 200 pollen grains per individual. The highest difference for the E value was found for shrub no. 2 (maximal value was 7.3% higher than the mean value) and for shrub no. 8 (minimal value was 12.9% lower than the mean value; Table 4). At the population level, the difference between the minimal value of E (e.g. obtained based on four pollen grains per shrub; 72 pollen grains in total) and the mean value obtained based on all 3600 pollen grains was 1.1%. For 12 out of 18 shrubs studied, the differences in mean E values obtained based on four, eight, 12, 16, 20, 28, 40, 80, 120 and 160 pollen grains were lower than 5% of the mean value obtained based on 200 pollen grains per individual. However, for some specimens, the differences among mean values of pollen grain morphological features obtained based on variable sample sizes were statistically significant. For example, statistically significant differences among mean values of E were found only for four shrubs (out of 18) (Table 4). Moreover, sample size had a statistically significant influence on mean values of P/E ratio of shrubs no. 8 and 12 (Table 5). For example, when five randomly selected pollen grains were used to estimate the mean value of P at the population level, all means were within the range estimated based on mean P ( 5%) at the population level (e.g mm; Table 6). However, when 10

10 Palynology 65 Figure 5. Boxplot of the relationship between values of equatorial diameter (E, mm) and analysed pollen samples size. Description as in Figure 4. randomly selected pollen grains were used, two mean values were out of the range established at the population level (mean P values from drawings no. 9 and 10 equals mm, which were only slightly less than the lower limit of the range, i.e mm). When 20, 30 and 50 pollen grains were used, all mean P values were within the range of the mean population-level value 5%. In the case of E, mean value at the population level was mm (the 5% range: mm). Only four mean values were out of the range; however, they were very close to the lower and upper limits of the range (e.g , and mm). Taking into consideration mean coefficients of variation (all drawings irrespective of number of pollen grains used for analysis), the pollen grain features analysed can be arranged as follows (from least to most variable): P, 7.6%; E, 9.4%; P/E, 10.8%. The ranges of coefficients of variation of particular pollen grain morphological features are similar irrespective of pollen sample sizes used in the analysis (Table 6). For example, the coefficient of variation (CV) of P ranges from 4.4 to 10.8% when five randomly selected pollen grains were used for the analysis, from 4.5 to 8.6% when 10 pollen grains were used for the analysis, from 6.8 to 9.3% when 20 pollen grains were used for the analysis, from 6.0 to 9.8% when 30 pollen grains were used for the analysis and from 6.9 to 9.3% when 50 randomly selected pollen grains were used for the analysis. The highest ranges of CV (from 4.8 to 17.5%) were noticed for the P/E ratio (Table 6). 4. Discussion The complexity of the problem of sample size in the pollen morphological studies and a lack of clear guidelines becomes apparent when a more accurate review of the palynological literature concerning the representatives of the Rosaceae family is performed. With respect to the size of pollen samples, all papers can be divided into several groups. The first group includes studies both older and more recent whose authors do not give numbers of pollen grains measured (Erdtman 1952; Reitsma 1966; Teppner 1966; Eide 1981; Fedoronchuk & Savitsky 1987; Tahir 2005; Chung et al. 2010). The second group incorporates articles which provide only an approximate sample size or a range.

11 66 D. Wronska-Pilarek et al. Figure 6. Boxplot of the relationship between values of P E ratio and analysed pollen samples size. Description as in Figure 4. For example, Popek (1996), analyzing the genus Rosa, examined from several to a dozen pollen grains. In their studies on the genus Rubus in China, Li et al. (2001) measured pollen grains, while Polyakova & Gataulina (2008) measured pollens from the genus Spiraea. Vafadar et al. (2010), in their study of genus Amygdalus, measured about 20 pollen grains. In Zamani et al. (2010) for the genus Pyrus the sample comprised pollen grains, whereas in Fatemi et al. (2012) for selected species from the genus Rosa the sample comprised about pollen grains. Another group of papers are studies in which different numbers of pollen grains were measured for the analysis of quantitative features and for qualitative features. For example, Tomlik-Wyremblewska (1995, 2000) and Tomlik-Wyremblewska et al. (2004), studying the genus Rubus, measured pollen grains for quantitative features and 10 pollen grains for qualitative features. In Isik & D onmez s (2004) studies of three pomoid genera, measurements were based on pollen grains for quantitative features and 1 10 for qualitative ones. Also, in the case of D onmez s (2008) paper on the pollen morphology of some Crataegus taxa, pollen size was measured for pollen grains, whereas other measurements were made on 10 grains. In some experiments on Malus species, Nazeri Joneghani (2008) measured in each case 10 pollen grains. He also analysed the length of the polar axis and the equatorial diameter but did not provide the number of pollen grains measured for these features. Finally, there are also papers in which authors give precise numbers of pollen grains measured within each sample and so, for example, Evrenosoglu & Misirli (2009) measured 10 pollen grains for each studied cultivar of some Rosaceae fruit species. The same number of pollen grains was analysed by Hebda et al. (1988a, 1988b, 1990, 1991) and Hebda & Chinnappa (1990, 1994) in a series of papers concerning pollen morphology of genera from the Rosaceae family, while in the study by Zhou et al. (1999), samples comprised 15 pollen grains for each analysed species from 10 genera from the Rosaceae family. Wang et al. (2009) measured 20 pollen grains, studying 12 taxa from the Idaeobatus section of the genus Rubus. Samples in studies conducted by Bednorz et al (some Sorbus species) as well as by Wronska-Pilarek & Jagodzinski (2009; 2011), Wronska-Pilarek (2011), and Wronska-Pilarek et al. (2012, 2013), who investigated species from the genera Rosa, Rubus and Crataegus, comprised 30 pollen grains. On the other hand, Monasterio-Huelin &

12 Palynology 67 Table 3. Mean values of length of polar axis ( standard error, SE) of Rosa canina determined for 18 individuals studied based on variable pollen grain sample sizes. Pollen grains were selected from each of four flowers collected from each of the rose shrubs (1/4 1 pollen grain from each flower, 4 pollen grains from the individual; 2/8 2 pollen grains from each flower, 8 pollen grains from the individual, etc.). One-way analyses of variance (ANOVAs) were performed separately for each individual shrub to determine the differences among mean values of P when variable sample sizes were used. The same letters indicate a lack of statistically significant differences between analysed sample sizes according to Tukey s a posteriori test (p < 0.05). Tendency (%) shows the difference between maximal and minimal P values determined with variable pollen grain sample sizes and mean value calculated based on 50 pollen grains per flower and 200 pollen grains per individual. Sample size/number of pollen grains ANOVA Tendency (%) Shrub no. 1/4 2/8 3/12 4/16 5/20 7/28 10/40 20/80 30/120 40/160 50/200 F P Max:mean Min:mean (1.89) (0.96) (0.73) (0.68) (0.57) (0.48) (0.39) (0.23) (0.18) (0.15) (0.13) (1.71) (0.92) (0.78) (0.66) (0.57) (0.45) (0.36) (0.25) (0.20) (0.17) (0.15) (0.96) (0.80) (0.69) (0.55) (0.47) (0.40) (0.31) (0.23) (0.20) (0.18) (0.16) (1.29) (1.18) (0.83) (0.64) (0.65) (0.49) (0.37) (0.26) (0.21) (0.19) (0.16) (0.50) (0.33) (0.69) (0.64) (0.57) (0.49) (0.38) (0.30) (0.24) (0.20) (0.17) (1.29) (0.92) (0.83) (0.68) (0.59) (0.43) (0.37) (0.29) (0.23) (0.19) (0.17) (0.58) (0.84) (0.63) (0.52) (0.56) (0.45) (0.37) (0.30) (0.23) (0.21) (0.19) (0.50) (0.84) (0.59) (0.53) (0.46) (0.37) (0.28) (0.24) (0.19) (0.16) (0.14) (1.15) (0.75) (0.56) (0.42) (0.38) (0.36) (0.34) (0.26) (0.22) (0.18) (0.16) (0.96) (0.76) (0.52) (0.40) (0.37) (0.38) (0.28) (0.21) (0.19) (0.16) (0.14) (1.26) (0.76) (0.70) (0.63) (0.64) (0.53) (0.44) (0.28) (0.22) (0.18) (0.16) (0.96) (0.91) (0.73) (0.62) (0.53) (0.44) (0.38) (0.25) (0.19) (0.18) (0.16) (0.96) (0.59) (0.39) (0.34) (0.31) (0.36) (0.31) (0.20) (0.16) (0.14) (0.13) (0.82) (0.65) (0.46) (0.36) (0.34) (0.30) (0.25) (0.20) (0.16) (0.15) (0.13) (1.26) (0.65) (0.58) (0.58) (0.49) (0.39) (0.34) (0.21) (0.18) (0.16) (0.14) (continued)

13 68 D. Wronska-Pilarek et al. Table 3. (Continued ) Sample size/number of pollen grains ANOVA Tendency (%) Shrub no. 1/4 2/8 3/12 4/16 5/20 7/28 10/40 20/80 30/120 40/160 50/200 F P Max:mean Min:mean (2.45) (1.36) (0.99) (0.93) (0.79) (0.60) (0.48) (0.32) (0.25) (0.21) (0.19) (1.89) (1.00) (0.75) (0.57) (0.49) (0.42) (0.36) (0.26) (0.20) (0.17) (0.14) (1.15) (0.70) (0.55) (0.58) (0.51) (0.48) (0.39) (0.32) (0.25) (0.21) (0.18) Mean (0.32) (0.22) (0.17) (0.15) (0.14) (0.11) (0.09) (0.07) (0.05) (0.05) (0.04) No. of pollen grains Table 4. Mean values of equatorial diameter ( standard error, SE) of Rosa canina determined for 18 individuals studied based on variable pollen grain sample sizes. Analysis description same as in Table 3 Sample size/number of pollen grains ANOVA Tendency (%) Shrub no. 1/4 2/8 3/12 4/16 5/20 7/28 10/40 20/80 30/120 40/160 50/200 F P Max:mean Min:mean (0.50) (0.33) (0.66) (0.63) (0.53) (0.46) (0.42) (0.25) (0.19) (0.16) (0.14) A 30.63A 30.25A 29.81A 29.85A 29.32A 29.23A 29.01A 28.96A 28.82A 28.90A (1.00) (0.63) (0.68) (0.58) (0.46) (0.43) (0.34) (0.23) (0.18) (0.16) (0.14) (0.58) (0.59) (0.59) (0.60) (0.49) (0.42) (0.34) (0.22) (0.17) (0.15) (0.13) (1.41) (0.92) (0.73) (0.60) (0.53) (0.49) (0.37) (0.26) (0.20) (0.18) (0.16) (0.96) (0.91) (0.86) (0.67) (0.66) (0.50) (0.39) (0.28) (0.23) (0.20) (0.18) (0.82) (0.45) (0.76) (0.86) (0.80) (0.64) (0.52) (0.36) (0.27) (0.23) (0.19) (continued)

14 Palynology 69 Table 4. (Continued ) Sample size/number of pollen grains ANOVA Tendency (%) Shrub no. 1/4 2/8 3/12 4/16 5/20 7/28 10/40 20/80 30/120 40/160 50/200 F P Max:mean Min:mean (1.50) (1.12) (0.78) (0.83) (0.71) (0.61) (0.54) (0.35) (0.26) (0.22) (0.20) BCD 25.25D 26.33CD 27.00ABCD 27.20ABCD 27.79ABCD 28.30ABC 28.55AB 28.62AB 28.61AB 28.69A < (1.00) (0.75) (0.73) (0.68) (0.59) (0.48) (0.42) (0.26) (0.20) (0.16) (0.15) (1.26) (0.80) (0.59) (0.51) (0.55) (0.58) (0.48) (0.36) (0.29) (0.25) (0.22) (1.71) (1.16) (0.94) (0.75) (0.62) (0.54) (0.42) (0.31) (0.27) (0.23) (0.21) (1.41) (1.28) (1.04) (0.88) (0.81) (0.62) (0.50) (0.32) (0.24) (0.20) (0.19) A 26.25A 27.33A 27.75A 27.70A 27.29A 26.85A 26.73A 26.23A 26.18A 26.04A (0.82) (0.70) (0.67) (0.68) (0.59) (0.52) (0.46) (0.29) (0.25) (0.21) (0.19) (1.41) (0.73) (0.52) (0.47) (0.45) (0.38) (0.35) (0.26) (0.21) (0.18) (0.17) (1.41) (0.70) (0.63) (0.51) (0.47) (0.39) (0.37) (0.23) (0.20) (0.17) (0.15) (0.96) (0.73) (0.52) (0.47) (0.41) (0.36) (0.28) (0.21) (0.17) (0.15) (0.14) A 26.00A 26.33A 26.38A 26.50A 26.93A 26.95A 27.10A 27.12A 27.19A 27.24A (1.71) (1.07) (0.77) (0.61) (0.50) (0.40) (0.30) (0.18) (0.15) (0.13) (0.11) (1.00) (0.53) (0.38) (0.35) (0.34) (0.31) (0.27) (0.21) (0.16) (0.14) (0.12) (0.50) (1.00) (0.67) (0.57) (0.56) (0.45) (0.35) (0.23) (0.18) (0.15) (0.13) Mean (0.33) (0.22) (0.19) (0.17) (0.15) (0.13) (0.10) (0.07) (0.06) (0.05) (0.04) No. of pollen grains ANOVA, analysis of variance.

15 70 D. Wronska-Pilarek et al. Table 5. Mean values of P/E ratio ( standard error, SE) of Rosa canina determined for 18 individuals studied based on variable pollen grain sample sizes. Analysis description same as in Table 3. Sample size number of pollen grains ANOVA Tendency (%) Shrub no. 1/4 2/8 3/12 4/16 5/20 7/28 10/40 20/80 30/120 40/160 50/200 F P Max:Mean Min:Mean (0.093) (0.046) (0.038) (0.035) (0.029) (0.024) (0.020) (0.012) (0.009) (0.008) (0.007) (0.029) (0.025) (0.020) (0.022) (0.019) (0.017) (0.014) (0.010) (0.008) (0.008) (0.007) (0.045) (0.038) (0.027) (0.032) (0.027) (0.024) (0.019) (0.011) (0.009) (0.008) (0.007) (0.087) (0.046) (0.035) (0.028) (0.024) (0.023) (0.018) (0.013) (0.010) (0.009) (0.008) (0.044) (0.033) (0.028) (0.024) (0.021) (0.018) (0.015) (0.011) (0.010) (0.009) (0.008) (0.039) (0.036) (0.038) (0.055) (0.047) (0.037) (0.031) (0.019) (0.014) (0.012) (0.010) (0.091) (0.071) (0.049) (0.049) (0.040) (0.032) (0.027) (0.018) (0.014) (0.011) (0.010) A 1.166AB 1.135AB 1.124AB 1.118AB 1.097AB 1.080AB 1.072AB 1.068B 1.067B 1.065B < (0.075) (0.051) (0.037) (0.029) (0.025) (0.020) (0.017) (0.012) (0.009) (0.007) (0.006) (0.041) (0.033) (0.024) (0.020) (0.021) (0.023) (0.019) (0.013) (0.012) (0.010) (0.009) (0.073) (0.051) (0.041) (0.033) (0.030) (0.026) (0.020) (0.015) (0.013) (0.011) (0.010) (0.093) (0.060) (0.056) (0.043) (0.035) (0.026) (0.021) (0.015) (0.011) (0.010) (0.009) A 1.206A 1.167A 1.157A 1.153A 1.159A 1.185A 1.189A 1.207A 1.215A 1.224A (0.052) (0.047) (0.041) (0.033) (0.029) (0.025) (0.022) (0.014) (0.012) (0.010) (0.009) (0.083) (0.045) (0.031) (0.029) (0.029) (0.026) (0.024) (0.015) (0.012) (0.009) (0.009) (0.042) (0.028) (0.026) (0.022) (0.023) (0.018) (0.015) (0.010) (0.009) (0.008) (0.007) (0.020) (0.031) (0.031) (0.026) (0.021) (0.018) (0.014) (0.010) (0.008) (0.007) (0.006) (0.070) (0.038) (0.031) (0.034) (0.028) (0.022) (0.019) (0.013) (0.010) (0.009) (0.008) (0.028) (0.023) (0.018) (0.019) (0.020) (0.019) (0.016) (0.011) (0.009) (0.008) (0.007) (continued)

16 Palynology 71 Table 5. (Continued ) Sample size number of pollen grains ANOVA Tendency (%) Shrub no. 1/4 2/8 3/12 4/16 5/20 7/28 10/40 20/80 30/120 40/160 50/200 F P Max:Mean Min:Mean (0.036) (0.043) (0.033) (0.029) (0.030) (0.025) (0.019) (0.013) (0.011) (0.009) (0.008) Mean (0.015) (0.010) (0.008) (0.008) (0.007) (0.006) (0.005) (0.003) (0.003) (0.002) (0.002) No of pollen grains Table 6. Mean values and coefficients of variation (CV) of the pollen grain morphological features determined based on 5, 10, 20, 30 and 50 randomly selected pollen grains of Rosa canina. Mean value of each morphological feature of pollen grains at the population level was calculated based on all 3600 pollen grains. Pollen grains used in the analysis were randomly selected from the database. Bold values indicate means which are lower/higher than 5% of mean value determined on population level. Drawing no. Feature Pollen grains no. Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean value on population level P(mm) E(mm) (continued)

17 72 D. Wronska-Pilarek et al. Table 6. (Continued ) Drawing no Mean value on population level Pollen grains no. Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Feature P/E Pardo (1995) measured 50 pollen grains in each sample collected for some species from the genus Rubus. Pipino et al. (2011) as well as Naruhashi & Takano (1980) worked on large samples of 400 and pollen grains, respectively. The former researchers investigated hybrid tea roses, while the latter authors studied size variation of pollen grains of four Rubus species. The results of this investigation are quite surprising. It turns out that at the level of a single Rosa canina flower as well as at the level of the specimen (shrub) or even the population, in order to obtain a mean value ( 5%) of the examined P and E features and P/E ratio, it is quite sufficient to measure more than five pollen grains or, according to some tests (Figures 1 3), more than 10 grains. This means, in practical terms, that the size of a pollen sample essential to secure a correct description of pollen grain morphology for the species examined and for the most important quantitative features (P and E) is smaller than would have been expected. As mentioned earlier, in the majority of recent palynological studies, samples comprise from 20 to 30 pollen grains. In the light of the results presented here, this value is sufficient for the correct description of quantitative pollen grain features, i.e. for the determination of the representative mean values of these features. However, it should be stressed that in order to acquire information regarding variability of the examined features at the population level, 30 pollen grains is a sufficient number, provided that they have been collected from at least several specimens (Tables 1, 3 6). Therefore, a sample containing 30 pollen grains, in general considered optimal for statistical studies, can also be recommended for palynological investigations because it yields a fully reliable result, i.e. the value of the given features of pollen grains is similar to the mean. It is also important to note that such relatively large pollen grain sample sizes make it possible to obtain more precise value ranges (min max) of the quantitative features analysed and, therefore, allow a more accurate pollen grain description of a given taxon. It is worth emphasising here that the plant material for palynological studies in the form of flowers or inflorescences is frequently derived from herbaria and, in such situations, we usually have one or at best several flowers collected from one specimen. Most frequently, it is not possible to find herbarium flowers derived from several plants belonging to the same population. The results obtained in this paper are not universal because they concern only one species (Rosa canina L.). Nevertheless, due to small variability of the analysed pollen features (P, E, P/E ratio) as well as to the structural similarity of pollen grains, they represent many genera from the Rosaceae family, e.g. Sorbus

18 Palynology 73 (Bednorz et al. 2005), Malus (Nazeri Joneghani 2008), Pyrus (Zamani et al. 2010), Rosa (Wronska-Pilarek & Jagodzinski 2011) or Crataegus (Wronska-Pilarek et al. 2013). Thus, we plan to determine how the results obtained in this paper can be applied to other numerous representatives of the colporate pollen class as well as other pollen classes. biology and genetics. Jan has authored or co-authored around 200 scientific publications. MAGDALENA JANYSZEK graduated with a PhD in horticulture during She is a researcher specialising on the systematics and ecology of the genus Carex L. (Cyperaceae). Currently, Magdalena s research has focussed on the carpology of sedges in terms of taxonomy, and the use of ornamental sedge seed heads as bedding plants. 5. Conclusions The results presented here indicate that if the exclusive objective of a palynologist is morphological description of pollen grains derived from one or several flowers of a specimen of a given species on the basis of quantitative features, the sample size should be higher than five and, according to other tests, more than 10 pollen grains. For reliability reasons, the higher sample size is recommended so, in practice, grains should be measured. In the case of studies on pollen grain variability, it is very important to refer the pollen size sample to the optimal sample size of 30 recommended in statistical studies. Tests demonstrated that such samples yield fully reliable results, which means that the obtained value of a given feature does not differ statistically from its mean value at the population level. Bearing in mind the above remarks, it can be said that the universal pollen sample size which can be applied in biometric studies in the case of pollen grain variability analysis is a sample comprising 30 pollen grains. Acknowledgements We kindly thank Dr. Lee E. Frelich (University of Minnesota, USA) for linguistic support and valuable comments on an early draft of the manuscript. We would like to thank the two anonymous reviewers for their detailed and valuable comments on the manuscript. Author biographies DOROTA WRO NSKA-PILAREK received her PhD in forestry during Her main research area is palynology, specifically the morphology and variability of pollen from the families Alliaceae, Cyperaceae, Fagaceae, Primulaceae and Rosaceae. Dorota also works on carpology, floristics and phytosociology. ANDRZEJ M. JAGODZI NSKI was awarded his PhD in He is a research scientist interested in identifying patterns of forest biodiversity and plant productivity of nondisturbed and degraded communities. He also works on plant ecology as a response to various environmental conditions. JAN BOCIANOWSKI has an MSc in mathematics awarded in 1998; he achieved a PhD in agriculture six years later in He is an interdisciplinary researcher, specialising in biometry and statistics for agriculture, plant breeding, References Alwadie HM Pollen morphology of six aquatic angiosperms from Saudi Arabia. Asian J Biol Sci. 1: Bednorz L, Maciejewska-Rutkowska I, Wronska-Pilarek D, Fujiki T Pollen morphology of Polish species of the genus Sorbus L. Acta Soc Bot Pol. 74: Borges RLB, Santos F, Giulietti AM Comparative pollen morphology and taxonomic considerations in Eriocaulaceae. 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