Pollen morphology and variability of species from the genus Rubus L. (Rosaceae) alien and invasive in Poland

Rubus L. is one of the most species-rich and most taxonomically challenging genera in the family Rosaceae. The aim of this study was to investigate pollen morphology and the ranges of interand intraspecific variability of the studied Rubus alien species, as well as verify the taxonomic usefulness of these traits in distinguishing studied taxa from this genus. We analysed six quantitative pollen characteristics and the following qualitative ones: exine ornamentation, pollen outline and shape. The study was conducted on 24 samples of six alien Rubus species found in Poland. 720 pollen grains were measured in total. The most important pollen features included exine ornamentation and length of the polar axis (P). In the conducted studies there were no features of pollen indicating “invasiveness”. The results of our research fill the gap in knowledge on pollen structure in Rubus species alien to Poland and Europe. They may constitute a foundation for further research on the reproduction of these species (e.g. pollen viability and fertility), thus facilitating identification of features determining their expansive character.


INTRODUCTION
Many bramble species are found outside their natural distribution area as a result of their use in horticulture, while some have been introduced accidentally (Király 2018). Alien brambles can become established very far from their original range and some of them became invasive (Evans and Weber 2003;Henderson 2007;Bennett et al. 2011;Clark et al. 2013). The identification of the taxa in the secondary range is difficult, especially in the case of apomictic species of Eurasian origin, with misidentifications, even using molecular methods, being relatively frequent (Alice et al. 2014;Bruckart et al. 2017). Király (2018) expressed an opinion that alien species from the genus Rubus generate significant ecological and nature conservational threats worldwide; however, the European flora and vegetation are not seriously affected by their impacts. Lambdon et al. (2008) reported that in Europe 35 Rubus species are 'aliens in Europe', 10 species are 'aliens of European origin' and 21 species -'aliens to Europe'. According to DAISIE (2009), in Europe 34 alien Rubus taxa are found, including all the studied species. Kurtto et al. (2010) reported in Europe 10 "non-native" Rubus species, and about 20 native species with secondary occurrences outside the original range. In turn, Tokarska- Guzik et al. (2012) showed for Poland the occurrence of six alien bramble species.
Rubus is one of the most taxonomically challenging genera in the family Rosaceae. Difficulties arise from the large number of species with similar morphological characteristics and often small, local ranges of natural occurrence, as well as polyploid hybridisation and apparently frequent facultative apomixis (Weber 1996;Alice and Campbell 1999). Therefore, among brambles, pollen of only 48 European species had been characterised until 2019 (Erdtman et al. 1961;Reitsma 1966;Teppner 1966;Eide 1981aEide , 1981bGonzalez Romano and Candau 1989;Monasterio-Huelin and Pardo 1995;Tomlik-Wyremblewska 1995;Wrońska-Pilarek et al. 2012, 2016Kosiński et al. 2018). These data have been supplemented by the latest comprehensive palynological study by Lechowicz et al. (2020), who described pollen grains of 58 Polish and European Rubus species, and by Xiong et al. (2019), who examined 155 species and 13 varieties representing all the 12 subgenera of this genus.
The most important features of bramble pollen grains include exine ornamentation (ornamentation type, width and orientation of striae and grooves), length of colpori, type of the bridge, costae colpi and the number and size of perforations (Reitsma 1966;Naruhashi and Takano 1980;Eide 1981aEide , 1981bKosenko et al. 1982;Gonzalez Romano and Candau 1989;Chinnappa 1990, 1994;Ueda 1992;Monasterio-Huelin and Pardo 1995;Tomlik-Wyremblewska 1995, 2000Li et al. 2001;Tomlik-Wyremblewska et al. 2004;Wrońska-Pilarek et al. 2006Wang et al. 2007;Kasalkheh et al. 2017;Gupta and Dash 2018;Xiong et al. 2019;Lechowicz et al. 2020). In the opinion of Tomlik-Wyremblewska (1995, 2000, pollen size and shape prove to be poor criteria in species identification. The aim of this study was to describe and analysed pollen morphology and the inter-and intraspecific variability of all six alien Rubus species (on the basis of 24 pollen samples) found in Poland, based on all pollen features. All of the studied bramble species, are distributed throughout Europe as aliens. We also investigated if pollen grains of the invasive and expansive bramble species differ in structure from pollen of other species, and whether any differences may affect their invasive properties.

Pollen morphology
The plant material was collected in the herbarium of the Institute of Dendrology, Polish Academy of Sciences in Kórnik (KOR) and stored in the herbarium of the Department of Forest Botany, the Poznań University of Life Sciences (PZNF), which did not require any permits to conduct research.
The study was conducted on 24 samples of six alien Rubus species (R. allegheniensis Porter, R. armeniacus Focke, R. canadensis L., R. laciniatus Willd., R. odoratus L. and R. xanthocarpus Bureau & Franch) found in Poland, the Czech Republic, Ukraine, Germany and France. A list of the species analysed with their affiliation to series is shown in Table 1.
In this paper the taxonomic classification of the studied taxa from the genus Rubus was adopted after Zieliński (2004), with further modifications (Kosiński et al. 2018). The verification of the taxa was made by Prof. Jerzy Zieliński (Institute of Dendrology, Polish Academy of Sciences in Kórnik), a batologist -taxonomist specialising in the genus Rubus.
Several, randomly selected inflorescences (flowers) were collected from 24 localities representing invasive Rubus species in five European countries (Table 2). Pollen grains were acetolysed according to the method of Erdtman (1960). The pollen grain samples were then mixed with 96% alcohol and centrifuged 4 times, with processed grains subsequently divided into two groups. One half of the processed sample was immersed in an alcohol-based solution of glycerin for LM, while the other was placed in 96% ethyl alcohol in preparation for scanning electron microscopy (SEM). The SEM observations were carried out using a Zeiss Evo 40 microscope, while LM measurements of acetolysed pollen grains were conducted at a 400× magnification using a Levenhuk D870T microscope equipped with a camera and software enabling accurate grain measurement. The exine sculptural elements were measured when taking SEM photos on an area of 25 μm 2 according to the methods of Ueda and Tomita (1989). SEM pictures were taken from samples numbered 1-5 (see: Table 2). Measurements taken from 30 mature, randomly selected, properly developed pollen grains were made using the light microscopy (LM), with 720 pollen grains measured in total. The pollen grains were analysed for six quantitative characters: length of the polar axis (P) and equatorial diameter (E), length of the ectoaperture (Le), distance between apices of two ectocolpi (d), and the P/E, and Le/P ratios ( Figure 1). The pollen shape classes (P/E ratio) were adopted according to the classification proposed by Erdtman (1952): oblate-spheroidal (0.89-0.99), spheroidal (1.00), prolate-spheroidal (1.01-1.14), subprolate (1.15-1.33) and prolate (1.34-2.00). In addition, the following qualitative characters were also determined: outline, shape, operculum structure and exine ornamentation.
Exine ornamentation types were identified based on the classification proposed by Ueda (1992). The types and subtypes of the striate exine ornamentation were characterised by the height and width of grooves, width of striae, and the number and diameter of perforations.

Statistical analysis
The normality of distributions for the studied traits (P, Le, d, E, P/E and Le/P) was tested using Shapiro-Wilk's normality test (Shapiro and Wilk 1965). Multivariate analysis of variance (MANOVA) was performed on the basis of the following model using a MANOVA procedure in the GenStat 18th edition: Y=XT+E, where: Y is the (n×p)-dimensional matrix of observations, n is the number of all observations, p is the number of traits (in this study p=6), X is the (n×k)-dimensional matrix of design, k is the number of species (in this study k=24), T is the (k×p)-dimensional matrix of unknown effects, and E -is the (n×p)-dimensional matrix of residuals. Next, one-way analysis of variance (one-way ANOVA) was carried out to determine the effects of Rubus samples on variability of P, Le, d, E, P/E and Le/P. The minimal, maximal and mean values as well as standard deviations of traits were calculated. Fisher's least significant differences (LSDs) were calculated for individual traits and on this basis homogeneous groups were established. The relationships between observed traits were assessed on the basis of Pearson's correlation. Relationships of six observed traits were presented in a heatmap. Results were also analysed using multivariate methods. The canonical variate analysis was applied in order to present multitrait assessment of similarity of tested samples in a lower number of dimensions with the least possible loss of information (Rencher 1992). This makes it possible to illustrate variation in species in terms of all observed traits in the graphic form. The Mahalanobis distance (Mahalanobis 1936) was suggested as a measure of "polytrait" species similarity (Seidler-Łożykowska and Bocianowski 2012), which significance was verified by means of critical value D α called "the least significant distance" (Camussi et al. 1985). Mahalanobis distances were calculated for all species samples. The differences between the analysed species were verified by cluster analysis using the nearest neighbour method and Euclidean distances. All the analyses were conducted using the GenStat 18th edition statistical software package.

General morphological description of pollen
A description of pollen grain morphology of the Rubus species studied is given below and illustrated in SEM photographs (Figures 2 and 3). Morphological observations for the other quantitative characters of pollen grains are shown in Tables 3 and 4.
Pollen grains of the Rubus species studied were tricolporate, isopolar monads ( Figure 2). According to the pollen size classification by Erdtman (1952) based on the length of the polar axis, analysed pollen grains were small (10-25 µm; 54.5%) or medium-sized (25.1-50 µm; 45.5%). Pollen grains had a small range of average values for trait P, ranging from 20.57 to 27.82 μm.
The average length of the polar axis (P) was 24.75 (15.16-33.65) µm (Table 3). The smallest mean P was found for pollen of R. xanthocarpus (20.57 µm), while the largest -for R. armeniacus (27.82 µm) (Table 3). In the R. xanthocarpus sample all measured pollen grains were small at a narrow range of polar axis length (18-24  µm). On the other hand, the longest pollen grains were found in R. armeniacus (22.19-31.45 µm).
The outline in the polar view was mostly circular with obtuse apices, less often elliptic, whereas in the equatorial view the outline was mostly elliptic, rarely circular ( Figure 2).
The polar area index (PAI) or the apocolpium index, in other words the d/E ratio, averaged 0.18 (0.05-0.41). The lowest mean value of this ratio (0.05) was recorded in R. laciniatus, while the highest (0.41) -in R. allegheniensis.
Rubus pollen grains usually have three apertures colpori. The colpori were arranged meridionally, regularly, more or less evenly spaced and were usually long, with a mean length of 20.34 (14.00-29.27) µm (Table 3). On average, the shortest colpori were found in R. xanthocarpus and R. canadensis (14.00 μm), while the longest was recorded in R. armeniacus (19.56 μm). On average, the length of the colporus (Le) constituted 82% of the polar axis length (P) and ranged from 67 to 97%. The colpori were narrow, linear or fusiform in outline. Their width varied and was usually greatest in the equatorial region. Sculpturing of ectocolpus membranes approached rugulate, rarely partly psilate (Figure 3). Colpus margins frequently had small undulations (Figure 3). In all the studied species a bridge was observed, crossing the colpus at the equator and dividing it into two parts, formed by two bulges of the ectexine that meet in the middle (Figure 3). These equatorial extensions were of the same or unequal length. Exine ornamentation in all species was striate-perforate, with the exception of R. odoratus, which had a verrucate ornamentation with small perforations ( Figure  3). Exine ornamentation elements were highly variable (Figure 3). Striae and grooves usually ran parallel to colpori and the polar axis, but frequently they also formed fingerprint-like twists. Striae were straight or forked and of varying length, width and height.
The investigated pollen of individual Rubus species was classified according to the striate exine ornamentation classification proposed by Ueda (1992). The cited author distinguished six types (I-VI) and six subtypes (I-III, each A and B). In our study only subtypes IIA and IIIA were found (Figure 3). Subtype IIA had prominent perforations between ridges and with short intervals of ridges. Subtype IIIA had more distinct ridges due to their steep slope; the ridges had small diameter (0.15~0.30 μm). Four species (R. allegheniensis, R. armeniacus, R. xanthocarpus, R. laciniatus) belonged to the IIA subtype, which was characterised by distinct striae, narrow grooves and frequently by prominent, numerous perforations. Subtype IIIA was represented by only one species (R. canadensis) with higher (i.e. more conspicuous than in subtype IIA), fairly narrow striae in comparison with subtype IIA. Striate-verrucate exine ornamentation with flattened and wavy muri and small, few perforations was found only in R. odoratus.

Interspecific variability of pollen grains
Results of MANOVA indicated that the all the Rubus samples were significantly different when investigated in terms of all the six quantitative traits jointly  (Tables 3 and 4). The mean values and standard deviations for the observed traits indicated a high variability among the tested samples, for which significant differences were found in terms of all the analysed morphological traits (Tables 3 and 4 The performed correlation analysis indicated statistically significant correlation coefficients for six out of 15 coefficients (Figure 6). Trait P was significantly correlated with Le (r=0.969), d (0.508) and E (0.955). Additionally, E was correlated with Le (0.915) and d (0.551). A negative significant correlation was observed between d and Le/P (-0.615).
In the presented dendrogram, as a result of the nearest neighbour grouping using the Euclidean distances method, the Rubus samples were divided into two groups (Figure 7). The first group comprised 14 samples, while the other was composed of the other 10 samples. In the dendrogram (Figure 7) a trend towards clustering of samples of individual species is observed, indicating similar traits of their pollen grains. The first group comprised almost all samples of R. laciniatus (samples 1-4), R. armeniacus (samples 1-5), and R. allegheniensis (samples 1-4), while the other group was composed of samples of R. canadensis and R. odoratus (Figure 7).
Individual traits vary in terms of their importance and share in the joint multivariate variation. A study on the multivariate variation for Rubus species samples includes also identification of the most important traits in the multivariate variation of species. Analysis of canonical variables is a statistical tool making it possible to solve the problem of multivariate relationships (Seidler-Łożykowska et al. 2013, Lahuta et al. 2018, Wrońska-Pilarek et al. 2018, Bocianowski and Majchrzak 2019. Figure 8 shows vari- ability of the six pollen grain traits in 24 studied Rubus samples in terms of the first two canonical variables. In the graph the coordinates of the point for particular species are the values for the first and second canonical variables, respectively. The first two canonical variables    accounted for 88.71% of the total multivariate variability among the individual species samples (Table 5, Figure 8). The most significant, positive, linear relationship between the first canonical variables was found for P, Le, d and E (Table 5). The second canonical variable was significantly positively correlated with d and negatively correlated with Le/P ( Table 5). The greatest variation in terms of all the traits jointly (measured Mahalanobis distances) was found for R. allegheniensis (sample 2) and R. canadensis (sample 4), with the Mahalanobis distance between them amounting to 4.78 (Table 6). The greatest similarity (0.56) was found for R. armeniacus (sample 1) and R. armeniacus (sample 4). The Mahalanobis distance is unitless and scale-invariant, and takes into account the correlations of the data set. On the basis of Mahalanobis distances we identified samples showing greatest similarity for all observed traits jointly.

DISCUSSION
In the lists of alien species of Europe (Lambdon et al. 2008;DAISIE 2009;Kurtto et al. 2010), the stud-ied Rubus species have the status of 'aliens in Europe' and naturalised within the continent (R. allegheniensis, R. armeniacus, R. canadensis, R odoratus, R. xanthocarpus) or 'aliens of European origin' -naturalised and neophytes within the continent (R. laciniatus). We studied all the six bramble species considered by Tokarska-Guzik et al. (2012) to be alien for Poland. All these species are domesticated kenophytes in Poland, occur-  ring naturally in North America and Asia; except for R. laciniatus -native to Eurasia, which in Poland is an invasive anthropophyte (www.iop.krakow.pl). In turn, R. xanthocarpus colonise anthropogenic plant communities, R. armeniacus -anthropogenic and seminatural ones, while the other four species are found in anthropogenic, seminatural as well as natural communities (Zieliński 1991;Bróź and Zieliński 1993;Weber 1993;Tokarska-Guzik et al. 2012). All palynologists agree that the most important pollen features for the species in the genus Rubus are the width, number and course of grooves (muri) and the width of the striae, as well as the number and diameter of perforations (Ueda and Tomita 1989;Ueda and Okada 1994;Tomlik-Wyremblewska 1995, 2000Li et al. 2001;Wrońska-Pilarek et al. 2006, Xiong et al. 2019Lechowicz et al. 2020). Some authors considered pollen size and shape as potentially important features in the diagnosis of the analysed Rubus species (Candau and Romanos 1987;Monasterio-Huelin and Pardo 1995;Wrońska-Pilarek et al. 2012;Ghosh and Saha 2017), while others claimed that they have no diagnostic significance (e.g. Li et al. 2001;Tomlik-Wyremblewska et al. 2004).
Based on Candau and Romanos (1987); Monasterio-Huelin and Pardo (1995); Wrońska-Pilarek et al. (2012) or Ghosh and Saha (2017) and our results, we partially agree with they former opinion, because the length of the polar axis (P) has been an important feature.
According to current palynological studies, European native bramble species are less variable in terms of exine ornamentation than Asian ones. Comprehensive palynological studies by Li et al. (2001), Xiong et al. (2019) and Lechowicz et al. (2020) confirmed this thesis. Li et al. (2001) examined 103 Rubus species from China, which belonged to four types and 11 subtypes, and Xiong et al. (2019) divided pollen grains of 168 taxa into six types and three subtypes of exine ornamentation. However, in the study of Lechowicz et al. (2020) in the examined 58 European Rubus species only two types of exine ornamentation were found (striate and striate-verrucate with microgranules). Other European palynologists distinguished in brambles mainly striate or striate-perforate exine ornamentation (Reitsma 1966;Eide 1981aEide , 1981bMonasterio-Huelin and Pardo 1995;Tomlik-Wyremblewska 1995, 2000Wrońska-Pilarek et al. 2006Polyakova and Gataulina 2008;Ghosh  and Saha 2017; Kasalkheh et al. 2017). Ueda and Tomita (1989) and Ueda (1992) distinguished six types and six subtypes of striate exine ornamentation in species from the genus Rosa and the family Rosaceae, including the genus Rubus. Hebda and Chinnappa (1990) divided Canadian rosaceous pollen types into two broad categories based on sculpturing. In the current study the analysed Rubus pollen grains were classified into two sculpturing subtypes (II A, III A), which were most frequently represented among the European native bramble pollen (e.g. Reitsma 1966;Eide 1981aEide , 1981bWrońska-Pilarek et al. 2012;Kasalkheh et al. 2017;Lechowicz et al. 2020).
Authors of many palynological studies (e.g. Tomlik-Wyremblewska 1995, 2000Li et al. 2001;Wrońska-Pilarek et al. 2012) reported that the bridges are located in most of the studied Rubus species. In a study by Lechowicz et al. (2020) and in our study, bridges were observed in all the analysed species. This structure was not used as a basis for the identification of species, because they were very similar in all species.
So far, morphological features of pollen grains have not been analysed in terms of their impact on the invasive character of the alien bramble species. Our results showed no differences in the pollen structure of the invasive R. laciniatus compared to the other five studied alien bramble species (Figures 7 and 8), but also with dozens of Rubus species native to Poland and Europe described by our team in another paper (Lechowicz et al. 2020). It may be stated that pollen morphology does not determine expansion of the studied species. Pollen viability and germination may have a greater impact on the expansive properties of such bramble species as R.
laciniatus. The arrangement of the investigated samples on the dendrogram (Figure 7) showed that samples of the same species (e.g. R. allegheniensis, R. armeniacus, R. odoratus) often form clusters, except for R. laciniatus. This means that R. laciniatus pollen grains were more similar to those of other species than to one another. CONCLUSIONS -Interspecific variability of pollen features was found with but the highest and the lowest variability observed for pollen of the same bramble species (e.g. R. allegheniensis -samples 1 and 3, R. laciniatussamples 4 and 5 or R. armeniacus -samples 5 and 2) (Figure7). -In the case of the invasive anthropophyte (R. laciniatus), the greatest intraspecific variability was found, as individuals of this species differed the most from each other (Figure 7), while no interspecies variation with other non-invasive bramble species was found. Thus so it may be assumed that pollen morphology probably does not affect invasive traits of this species. -Results of our studies fill the gap in knowledge on pollen structure of species from the genus Rubus alien for Poland and Europe. They may be used in further studies on reproduction of these species (e.g. pollen viability and fertility), facilitating identification of traits determining their expansive character.