untitled 74 ACTA BOT. CROAT. 75 (1), 2016 Acta Bot. Croat. 75 (1), 74–80, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0009 ISSN 0365-0588 eISSN 1847-8476 Morphology of Anemone sylvestris L. fl ower (Ranunculaceae) Bożena Denisow1*, Sebastian Antoń1, Małgorzata Wrzesień2 1 Department of Botany, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland 2 Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland Abstract – During the monitoring of populations of Anemone sylvestris L. (Ranunculaceae), a protected spe- cies in Poland, we found that the seed set is impaired. The fl ower is considered an adaptation that has co- evolved to achieve effective pollination and successful fertilization. Therefore we have focused on the mor- phological and anatomical characteristics of the fl owers of A. sylvestris L. as a prelude to the study of the species’ pollination biology and plant breeding system. The large size of the fl ower (50.6 ± 16.4 mm in di- mensions) and its bowl shape fulfi l both the biotic pollination syndrome and the aerodynamic requirements for pollen dispersal and capture. The opening and closing of the perianth provide a shelter for beetles. The odourless perianth, absence of nectar, scarcity of pollen (approximately 200 000 pollen grains per fl ower) and its traits – small size (axis P = 18.52 ± 1.0 μm; E = 16.59 ± 0.9 μm), lack of balsam on the exine surface, starch accumulation in more than 95% of pollen grains correspond to the specialization in anemophily. The stigma is papillous, the dense hairs are situated between single carpels indicating adaptation to capturing dry pollen and specialization in the wind pollination syndrome. The fl ower of A. sylvestris is an example for an intermediate form between entomophily and anemophily, i.e. a secondary and more advanced feature among Ranunculaceae. Keywords: androecium, Anemone sylvestris, anther, gynoecium, papille, pollen, Ranunculaceae, trichomes * Corresponding author, e-mail: bozena.denisow@up.lublin.pl Introduction Different interdependent factors may infl uence the effi - ciency of pollen dispersal between individuals, i.e. density of population or pollen vectors (Nishikawa and Kudo 1995, Aizen and Harder 2007). The most signifi cant adaptation that co-evolved in order to maximize pollination is that based on fl oral morphology (Faegri and Van Der Pijl 1979, Konarska 2014, Sulborska et al. 2014). Typically, colour, shape, scent, display size, and symmetry start the reaction chain of direct or indirect action that leads to pollination (Willmer 2011). However, some fl oral structures, e.g. nec- taries, characteristics of the style and stamens, or pollen traits have been recognized as fundamental traits of special- ization in the pollination syndrome (e.g. anemophily vs. en- tomophily vs. self-pollination), although numerous excep- tions from typical adaptation exist (Friedman and Barrett 2009). The Ranunculaceae is a family with 59 genera and is considered the most primitive of angiosperms (Tamura 1995), as well as one of the basic groups of eudicots in APG III (Chase and Reveal 2009). Within this botanical family, diverse evolutionary tendencies in the fl ower mor- phology and pollination syndrome occur (Endress 1995, Tamura 1995, Willmer 2011). Some genera, i.e. Ranuncu- lus, Hepatica, Adonis, or Ficaria possess fl owers with mor- phological traits fulfi lling the requirements of generalist in- sect pollinators (Denisow et al. 2014). The species that develop spurs or pockets from the perianth and share nec- taries contribute to the specialized insect pollination syn- drome, e.g. Aconitum or Aquilegia (Endress 1995, Denisow and Antoń 2012, Antoń and Denisow 2014), whereas others are adapted to wind pollination, e.g. species from the genus Thalictrum (Friedman and Barrett 2009). A combination of insect and wind pollination has been reported for some spe- cies from the genus Clematis (Tamura 1995). The genus Anemone L. s. str. (Ranunculaceae) is closely related to Hepatica, Clematis, Anemonella, and Ranunculus (Chase and Reveal 2009). The Anemone species are distrib- uted primarily in the northern temperate zones (Hegi 1974, Tamura 1995). In Europe, approximately 17 species from four different subgenera may be found in natural popula- tions (Müller 2002, Ziman et al. 2011). Four species are na- tive to Polish fl ora; they occur in different habitats, i.e. de- FLOWER MORPHOLOGY OF ANEMONE SYLVESTRIS ACTA BOT. CROAT. 75 (1), 2016 75 ciduous forests, xerothermic grasslands, or in mountain fl ora (Zając and Zając 2009). Flowers from the genus Anemone were recognized as nectarless and exclusively polleniferous (Horovitz 1991, Nishikawa and Kudo 1995, Szklanowska 1995, Denisow and Bożek 2006); however, nectar production has been documented in Anemone nemorosa (Erbar and Leins 2013). Generally, fl owers devoid of nectaries are considered less specialized with regard to the pollination system, than fl ow- ers that do develop nectaries (Erbar et al. 1999). Anemone sylvestris L. is a perennial herb native to Eur- asia and distributed from Central Europe through the Cau- casus, South Siberia to the northwest regions of China (Hegi 1974). It is a common species in the Balkans (Alba- nia, Bosnia and Herzegovina, Bulgaria, Croatia, Greece, Macedonia, Montenegro, Serbia, and Slovenia), occurring in semi-shady steppe-forest communities (Zimman et al. 2011). In Northern Europe, A. sylvestris is found in xero- thermic grasslands of the Festuco-Brometea class and is a characteristic species for the Geranio-Anemonetum sylves- tris association or grows in thermophilous thickets of Cory- lus avellana (Piękoś-Mirkowa and Mirek 2006, Maciejews- ka-Rutkowska and Antkowiak 2013). In Poland, it is an endangered plant, protected by law (Piękoś-Mirkowa and Mirek 2006, Kwiatkowska-Falińska and Faliński 2007). Recovery programs for A. sylvestris primarily aim at con- servation and management of the habitat (Wrzesień and Denisow 2006, Chmura et al. 2013). It is a long-lived, rhi- zomatous clonal species. Individuals may produce several fl owers each year. The species also has a capability of sexu- al reproduction (Ehrendorfer et al. 2009). A number of stud- ies have pointed out that sexual reproduction is a prerequi- site for maintenance and development of sustainable population, even for clonal species, since it counteracts the inbreeding depression (Müller 2002). The monitoring of three wild populations of A. sylves- tris located in SE Poland conducted in short- and long-term perspective revealed their decrease in size (Denisow and Wrzesień 2015, in press). Vegetation changes, e.g. shrub encroachment, are possible explanations (Kwiatkowska- Falińska and Faliński 2007). In addition, there are problems with seed set, as we observed only 9–12% of seeds devel- oped in relation to one-ovuled pistils. In the present paper, we focused on the morphological characteristics of the fl owers of A. sylvestris L. as a prelude to the study of the species’ pollination biology and breeding system. In particular, we focused on (i) primary and sec- ondary fl oral attractants, (ii) pollen production and pollen traits. Materials and methods Flowering of Anemone sylvestris L. (Ranunculaceae) was observed in 2013 and 2014 in a population located in Stawska Góra (51°13’N, 23°25’E; 224.8 m a.s.l.) in the Lu- blin Upland, SE Poland. The A. sylvestris was a component of a loose grassland patch of Brachypodio-Teucrietum from the Festuco-Brometea class. Additionally, for microscopic examinations performed in 2014, we used fl owers from in- dividuals cultivated in the Botanical Garden of Maria Curie-Skłodowska University, Lublin, SE Poland (51°15’ 44’’N, 22°30’48’’E). The collection was established on the basis of individuals derived from the Stawska Góra popula- tion. Flower biology The duration of the fl ower life span was recorded in 2013 (n = 11 fl owers) and in 2014 (n = 15 fl owers). We de- fi ned the total fl ower lifetime as the period from fl ower opening to corolla shedding. To determine the temporal separation of stigma receptivity and anther dehiscence, the fl ower development was monitored from the bud stage until the end of pollen presentation. The number of anthers and the number of pistils per fl ower (n = 23 in 2013 and n = 27 in 2014) were established. Every day we recorded the de- gree to which stigmas were exerted as well as the number of dehiscing anthers. Stigma receptivity The timing of stigma receptivity was determined; 30% hydrogen peroxide was applied for detection of peroxidase activity (SPA) (Dafni 1992). The entire gynoecium from the fl owers was extracted and was subdivided for three parts (low, medial, apical). Each part was placed on a glass slide separately and coated with a drop of H2O2. Stigmas that produced bubbles within 2–3 min were considered re- ceptive. The number of receptive stigmas was counted for each individual fl ower (n = 9 fl owers per consecutive day of anthesis) under a binocular microscope (NIKON SMZ-2B). Flower size and micromorphology The fl ower size was established by means of fl ower di- ameter measurements (n = 20 fl owers) at the full fl owering stage. The length between the external points of the petal- oid sepals was measured. These measurements were per- formed using a digital calliper with an accuracy of 0.02 mm. The morphological and anatomical details were exam- ined by means of light microscopy (LM) and scanning elec- tron microscopy (SEM). The photographic documentation was made on freshly cut material using an Olympus SZX12 stereomicroscope equipped with a Canon EOS 550D digital camera. The material used for SEM was fi xed in 2.5% glu- taraldehyde in phosphate buffer (pH 7.4; 0.1 M) at a tem- perature of 4 °C for 12 hours. Next, the material was washed in phosphate buffer and dehydrated in graded ace- tone series, respectively. Afterwards, the plant material was critical-point dried using liquid CO2, sputter coated with gold, and examined at an accelerating voltage of 30 kV with a TESCAN/VEGA LMU scanning electron micro- scope. Pollen production and pollen characteristics The number of pollen grains per anther and fl ower and pollen grain size were determined. The anthers (n = 16 in 2013 and n = 20 in 2014) were dissected from closed fl ow- DENISOW B., ANTOŃ S., WRZESIEŃ M. 76 ACTA BOT. CROAT. 75 (1), 2016 ers (n = 11 in 2013 and n = 14 in 2014). Next, the anthers were placed on a microscopic glass, the pollen sacs were squashed, and the anther walls were carefully removed. Af- terwards, we put on a drop of aniline blue with glycerine and the number of pollen grains was counted. Pollen grain dimensions were determined in glycerol-gelatine slides (Erdtman 1954). The lengths of the polar axis (P) and the equatorial axis (E) were determined (n = 4 × 50 per year). These observations were conducted using a Nikon Eclipse 200 light microscope. The protein content was detected in dry samples collected during the study period. The Kjeldahl method was used for nitrogen content determination and crude protein was estimated using factor 6.25 (Roulston and Cane 2000). Starch accumulation was detected with the Lugol’s iodine solution in 200 pollen grains per year. Data analysis Standard ANOVA was applied to assess inter-year dif- ferences in the mean values of the analyzed criteria. In or- der to detect differences between the means, post hoc com- parison was made by means of the Tukey test. Data are presented as mean values ± standard deviation (SD). The level of statistical signifi cance required to measure differ- ences between the means for all the analyses was P = 0.05. All data analyses were performed using STATISTICA 10.0 (Statsoft Inc.) software. Results The fl owering of A. sylvestris lasted 4–6 weeks, with peak fl owering periods differing up to 4 weeks between the years (Tab. 1). The peak of the fl owering time of the species was recorded in April (2013) or in May (2014). The repro- ductive shoot is unbranched, 10–15 cm high. The fl ower is perfect, actinomorphic, and odour-less. The pentamerous sepals are white and obovate; the fl ower lacks petals (Figs. 1A–C). The mean dimension of the fl ower is 50.6 ± 16.4 mm (n = 20). Anthesis of a single fl ower typically lasted 4–6 days (n = 11–15). For the fi rst 2–3 days, the fl ower opened between 9.00 and 11.00 and closed at ca. 17.00. A temperature decrease (< 10 °C) kept the fl owers closed, or their closure was observed before 17.00. Beetles, e.g. Mordellistena sp. were observed in the fl owers. During the course of fl owering, sepal turgor gradually decreased, and from the third day of anthesis, the sepals in many fl owers were not able to open fully. When the fl ower opened the fi rst time, multiple stigmas were inserted over the top of the immature anthers. The fi rst anthers dehisced on day 3–4 af- ter the fi rst fl ower opening, and the remaining anthers de- hisced progressively during the successive days. The fl ower development of A. sylvestris allowed us to distinguish 5 relatively distinct stages: stage 1 – tight white- greenish bud, no stigma receptivity and no pollen release; stage 2 – petals closed, beginning of stigma receptivity; stage 3 – petals opened, stigma receptivity in > 30% of pis- tils, stage 4 – intermezzo – early male stage, dehiscence of the fi rst anthers, stigma receptivity; stage 5 – > 30% of an- thers had dehisced, aborted ovules, or developed achenes. Androecium and gynoecium characteristics The fl oral organs are free and arranged in a spiral se- quence on a convex receptacle (Figs. 1C–D). Nectariferous tissue is absent. The androecium consists of numerous sta- mens, ranging from 57 to 146 per fl ower (Tab. 1). The num- ber of stamens per fl ower varied signifi cantly from year to year (F1,11 = 19.7, P = 0.044). The anthers are ovoid-elon- gated. The anthers within the androecium dehisce gradual- ly, starting at the middle. Opening of the ripe anther is lon- gitudinal. Three-fold disparities in the number of pollen grains produced per anther were found to occur between the study seasons (F1,16 = 9.6, P = 0.034; Tab. 2). The gynoecium possesses numerous apocarpic carpels (Figs. 1C–D), whose number ranged from 94 to 184 per fl ower. The number of pistils per fl ower varied signifi cantly from year to year (F1,11 = 12.1, P = 0.032). Flower micromorphology The adaxial epidermis of the petaloid sepals is smooth (Fig. 2A), whereas the abaxial epidermis bears numerous, Tab. 1. Phenology and fl oral characteristics (mean ± standard de- viation, with range in parentheses) of Anemone sylvestris from natural population in Stawska Góra, SE Poland, during a 2 year study. Means with the same small letter do not differ signifi cantly between years at P < 0.05, based on HSD Tukey test. Feature 2013 2014 Flowering period 10 April – 10 May 26 April – 5 June Duration of fl owering (days) 31 55 No. of anthers per fl ower 103.5a ± 36.2 (87 – 131) (n = 23) 87.9b ± 23.1 (57 – 146) (n = 27) No. of pistils per fl ower 112.3a ± 26.8 (94 – 126) (n = 23) 157.6b ± 53.1 (108 – 184) (n = 27) Fig. 1. Macrophotographs of the of Anemone sylvestris: A) plants in the experimental population (bar = 10 cm); B) solitary, white fl owers (bar = 5 cm); C) spirally arranged, multi-staminate an- droecium (bar = 1 cm); D) longitudinal section through gynoeci- um showing numerous carpels (bar = 0.5 cm). FLOWER MORPHOLOGY OF ANEMONE SYLVESTRIS ACTA BOT. CROAT. 75 (1), 2016 77 long, unicellular hairs with the greatest density observed at the base (Figs. 2B, E). In cross sections, the petaloid sepals are relatively thin with 4–6 mesophyll layers and have re- duced vascular bundles (Fig. 2C). Numerous hairs between distinct apocarpic carpels are present (Fig. 1D; Figs. 2D–H). Each carpel contains a sin- gle ovule, which develops into a one-seeded achene. The receptacle enlarges after anthesis and continues to enlarge during achene formation in a fruit cluster of fruitlets. Stig- matic tissue is visible at the apex of the style (Figs. 2F–H). The stigmatic area is easily distinguishable from the style, bearing a number of unicellular and conical papillae, whereas the surface of the style is covered by smooth epi- dermal cells (Figs. 2H–I). The presented stigma is dry. The anther walls consist of the epidermis, endothecium, middle layer, and tapetum (Figs. 3A–D). The endothecium has thickenings that are differentiated in the lateral walls of the cells (Figs. 3C–D). The pollen grains are dry, tricolpate with a reticulate surface (Fig. 3E), and prolato-spheroid (shape index 1.09–1.13). The diameter of the polar axis (P) Fig. 3. Scanning electron (A–E) and light (F) micrographs of the anther and pollen grains of A. sylvestris: A) general aspect of the stamen (bar = 500 μm); B) longitudinal section through anther with visible connective (c) (bar = 200 μm); C–D) section through anther showing tapetum (arrow), endothecium cells with wall thicknesses (arrowhead) and numerous pollen grains (pg) (bars = 20 μm); E) tricolpate pollen grains (bar = 20 μm); f) pollen grains stained with Lugol’s iodine (bar = 10 μm). Fig. 2. Scanning electron micrographs of the fl oral organs of A. sylvestris: A) smooth adaxial surface of the petaloid-sepal (bar = 500 μm); B) abaxial surface of the petaloid-sepal with numerous hairs (bar = 500 μm);C) longitudinal section through petaloid-se- pal, note reduced vascular bundle (bar = 20 μm); D) section through gynoecium showing numerous pistils (bar = 500 μm); E) longitudinal section through fl ower, from the external to internal part: petaloid-sepals, stamens, pistil (bar = 200 μm); F, G) general aspect of the multiple gynoecium with distinct apocarpic carpels (bars = 200 μm and 500 μm, respectively); H) details of a single carpel with easily distinguishable stigmatic area (bar = 100 μm); I) stigmatic area of pistils with a number of unicellular and conical papillae (bar = 20 μm). Tab. 2. Pollen production and pollen grain characteristics (mean ± standard deviation, with range in parentheses) of Anemone sylvestris from natural population in Stawska Góra, SE Poland, during a 2 year study. Means with the same small letter do not differ signifi cantly between years at P < 0.05, based on Tukey test. Year No. of pollen grains Length of axis (μm) Shape index (P/E)per anther per fl ower polar (n =200) equatorial (n = 200) 2013 930.2a ± 484.0 (110 – 1790) (n = 16) 96275 (80927 – 121856) 18.31a ± 1.02 (16.56 – 19.68) 16.71a ± 0.61 (14.02 – 17.23) 1.09 2014 3361.0b ±825.3 (70 – 4480) (n = 20) 295432 (191577 – 490706) 18.73a ± 0.91 (17.34 – 20.88) 16.47a ± 0.85 (14.67 – 17.96) 1.13 Mean for years 2144.7 195853 18.52 ± 1.01 16.59 ± 0.92 1.11 DENISOW B., ANTOŃ S., WRZESIEŃ M. 78 ACTA BOT. CROAT. 75 (1), 2016 ranges from 16.56 to 20.88 μm and the equatorial axis (E) is in the range of 14.02–17.96 μm. No year effect on the mean values of the diameter of pollen axis was found (F1,6 = 2.3, P = 0.124 for polar and F1,6 = 1.2, P = 0.085 for equato- rial). Starch was present in more than 95% of pollen grains (Fig. 3F). The mean protein content in the pollen is 24.6% of dry matter ± 0.75. Discussion The morphological architecture and extrafl oral attri- butes of A. sylvestris fl owers imply that two pollination modes may interact to affect pollen dispersal. Secondary at- tractants, sensu Faegri and Van Der Pijl (1979), i.e. visual and/or temperature attractants, indicate that the fl owers of A. sylvestris may lure insects as potential pollen vectors. On the other hand, the specifi city of fl oral reward, the traits of pollen grains, as well as some gynoecium traits indicate in- volvement of wind in the dispersal of pollen. A. sylvestris fl owers are large and bowl-shaped, accord- ing to the classifi cation of Faegri and Van Der Pijl (1979). In general, this fl ower type seems to function as an advertis- ing organ for luring insect visitors (Willmer 2011); it also fulfi ls the aerodynamic requirements for pollen dispersal and capture (Friedman and Barrett 2009). The perianth is scentless and the feature corresponds to that in anemophi- lous taxa whose perianth is still visually attractive (Willmer 2011). From the third day of anthesis, the turgor of the sepals decreased, which apparently impaired sepal opening. We also observed closing of the fl ower for the night and during the day when temperature dropped below 10 °C. Closed fl owers prevent the wind from serving as a pollen dispersal agent. However, the anthers of A. sylvestris inserted on thin fi laments are likely to contact stigmas when the fl ower is blown by the wind; therefore, self-pollination is highly pos- sible. Closed fl owers are also temporarily unavailable for insects. We found beetles both in closed fl owers (presum- ably warming up or avoiding the wind) and in opened fl ow- ers on sunny days (presumably maximizing exposure to so- lar radiation in order to build up heat before take-off). The syndrome of the opening and closure of the fl ower, which provides a shelter for ectotherms, is referred to as a tem- perature attractant (Faegri and Van Der Pijl 1979) and has already been documented for several species, e.g. Adonis vernalis (Denisow et al. 2014), Anemone patens (Ordway 1986), A. canadensis (Douglas and Cruden 1994), or A. nemorosa (Van Doorn and Van Meeteren 2003). However, the signifi cance of insects that take shelter inside a fl ower is not obvious, and requires further experimental investiga- tions to establish their effi ciency in the pollination process, e.g. the number of pollen grains they actually transfer and deposit on the stigmas. In most species, perianth movements of a thermonastic type are associated with a difference in the growth rate of mesophyll cells (Van Doorn and Van Meeteren 2003). Pre- sumably, the relatively thin mesophyll layer of the petaloid sepals revealed in A. sylvestris optimizes the movement at a minimal metabolic cost. Among the noteworthy results of the current study is the fi nding that A. sylvestris fl owers lack nectar-secreting struc- tures. The attribute can be considered as promotion of ane- mophily (Friedman and Barrett 2009) and is interpreted as a co-evolutionary adaptation to attraction of specifi c type of insects (Waldbauer and Friedman 1991). Absence of nectar was confi rmed for other Anemone species, i.e. A. coronaria, A. canadensis, A. fl accida, or A. japonica, the fl owers of which were visited mainly by beetles; however, fl ies or primitive Apoidea (Anthophoridae, Halictidae) were also observed (Horovitz 1975, Horowitz 1991, Douglas and Cruden 1994, Nishikawa and Kudo 1995, Denisow and Bożek 2006). Conversely, nectar-producing fl owers of A. nemorosa were reported to be visited by Bombylius spp., honeybees, or bumblebees (Szklanowska 1995, Erbar and Leins 2013). Probably, the nectar defi ciency in the fl owers of A. sylvestris restricts the range of the insect spectrum, resulting in the scarcity of dipterans and bee visitors in our experimental population (manuscript in preparation). Not only was nectar absent, but a relatively small amount of pollen was produced in a single fl ower of A. syl- vestris. Altogether, the primary attractant characteristics possibly explain the low interest of insect visitors. Although the fl owers are multi-staminate, they released only approx. 200 000 pollen grains per fl ower. The value is ten-fold low- er than that established for Anemone coronaria (approx. two million). Generally, taxa from the family Ranunculace- ae are reported to produce copious amounts of pollen (Sz- klanowska 1995, Denisow and Bożek 2006, Denisow and Antoń 2012). The low pollen production in A. sylvestris may be related to the genetic potential of the species; pollen production is reported to be highly genetically dependent (Szklanowska 1995, Denisow 2011). However, the year-to- year disparities in the number of pollen grains produced in- dicate that pollen production in A. sylvestris is sensitive to external factors. For example, weather conditions (shortage of precipitation or air temperature anomalies) are known to limit pollen production in a number of taxa (Denisow and Bożek 2006, Aizen and Harder 2007, Denisow et al. 2014). The pollen grains of A. sylvestris are small, within a range of 10–25 μm (Erdtman 1954). The protein content of pollen is approximately 25% of the dry mass. The protein concentration in pollen grains is a highly conserved trait within plant genera and families; however, the correlation between the protein content and the type of pollination is not always obvious (Roulston and Cane 2000, Denisow 2011). Nonetheless, small pollen grains, lack of balsam on the exine surface, and starch accumulation correspond well to specialization in anemophily (Friedman and Barrett 2009). Similar pollen traits were described for Anemone coronaria by Horowitz (1991), who proved that pollen was dispersed by air currents up to the distance of < 1.5 m from the fl ower. We found papillous stigma and dense hairs situated be- tween single carpels. It is possible that these properties en- hance the capture of dry pollen and may indicate specializa- tion in the wind pollination syndrome, as suggested by Horowitz (1991). However, the pistils placed on the convex receptacle are well exposed and provide an unobstructed FLOWER MORPHOLOGY OF ANEMONE SYLVESTRIS ACTA BOT. CROAT. 75 (1), 2016 79 path for pollen transported by both abiotic and biotic agents (Friedman and Barrett 2009). The other ecologically signif- icant adjustment of these dense hairs seems to be protection of pistils and ovules from overheating and/or heat loss. Low temperatures are particularly frequent during early spring, and they are often hazardous for carpels, which are consid- ered the most sensitive plant structures (Hedhly 2011). The anthers of A. sylvestris dehisce longitudinally. We found lignifi ed wall thickenings in the endothecium cells. Many investigation results are consistent in showing that cell wall thickenings control the dynamics of anther opening by regulating the dehydration and shrinkage of cells, which may indirectly support pollen dispersal (Keijzer 1987). The abaxial surface of the petaloid sepals is velvety. It is associated with the presence of numerous hairs on the ab- axial epidermis. The same characteristic of abaxial epider- mis was evidenced in the fl owers of Adonis vernalis (Gos- tin 2009). Epidermal hairs are reported to protect epidermal and mesophyllous cells against excessive heating or water loss, which is especially important in xerothermic habitats (Karabourniotis et al. 1992). The adaptive signifi cance of epidermal hairs in the defence against phytophagous insects has also been confi rmed (Hanley et al. 2007). A. sylvestris fl owers are protogynous, which has been confi rmed by the fi ndings for other Anemone species (Horowitz 1991, Denisow and Bożek 2006). Protogyny is commonly described in cantharophilous fl owers (Faegri and Van Der Pijl 1979) as well as in anemophilous ones (Friedman and Barrett 2009). In the protogynous fl owers of A. sylvestris, the absence of nectar may affect the function of the female phase by decreasing the attractiveness of the phase to potential pollinators and infl uence the mating op- portunity of the fl ower. Presumably, the fl ower of A. sylvestris is an example of an intermediate form between entomophily and anemophi- ly, i.e. a secondary and more advanced feature among Ra- nunculaceae (Endress 1995). The fl oral morphology and the type of the fl oral attractant indicate the existence of alterna- tive modes of pollination (partial wind, self-pollination, or biotic pollination). Acknowledgements The material from protected species was collected in compliance with Polish law under permit from the Regional Nature Conservator in Lublin. The research was supported fi nancially by the Ministry of Science and Higher Educa- tion of Poland as a part of the statutory activities of the De- partment of Botany, University of Life Sciences in Lublin (project: OKB/DS/2) and the Department of Geobotany, In- stitute of Biology and Biochemistry, Maria Curie- Skłodowska University. References Aizen, M. A., Harder, L. D., 2007: Expanding the limits of the pollen limitation concept: effects of pollen quantity and quali- ty. Ecology 99, 271–281. Antoń, S., Denisow, B., 2014: Nectar production and carbohydrate composition across fl oral sexual phases: contrasting patterns in two protandrous Aconitum species (Delphinieae, Ranuncu- laceae). Flora 209, 464–470. Chase, M. W., Reveal, J. L., 2009: A phylogenetic classifi cation of the land plants to accompany APG III. Botanical Journal of the Linnean Society 161, 122–127. Chmura, D., Adamski, P., Denisiuk, Z., 2013: How do plant com- munities and fl ower visitors relate? A case study of semi-natu- ral xerothermis grasslands. Acta Societatis Botanicorum Po- loniae 82, 99–105. Dafni, A., 1992: Pollination ecology, a practical approach. Oxford University Press, Oxford. Denisow, B., Bożek, M., 2006: Blooming biology and pollen abundance of Anemone japonica Houtt. = Anemone x hybrida hort. Acta Agrobotanica 59, 139–146, (in Polish). Denisow, B., 2011: Pollen production of selected ruderal plant species in the Lublin area. University of Life Sciences in Lub- lin Press, 351. Denisow, B., Antoń, S., 2012: Flowering, nectar secretion, pollen shed and insect foraging on Aquilegia vulgaris L. (Ranuncula- ceae). Acta Agrobotanica 65, 37–44. Denisow, B., Wrzesień, M., Cwener, A., 2014: Pollination and fl o- ral biology of Adonis vernalis L. (Ranunculaceae) – a case study of threatened species. Acta Societatis Botanicorum Po- loniae 83, 29–37. Denisow, B., Wrzesień, M. 2015: Does vegetation impact on the population dynamics and male function in Anemone sylvestris L. (Ranunculaceae)? A case study in three natural populations of xerothermic grasslands. Acta Societatis Botanicorum Po- loniae DOI:10.5586/asbp.2015.017, (in press). Douglas, K. L., Cruden, R. W., 1994: The reproductive biology of Anemone canadensis (Ranunculaceae): breeding system and facilitation of sexual selection. American Journal of Botany 81, 314–321. Ehrendorfer, F., Ziman, S. N., König, C., Keener, C. S., Dutton, B. E., Tsarenko, O. N., Bulakh, E. V., Boşcaiu, M., Médail, F., Kästner, A., 2009: Taxonomic revision, phylogeny and trans- continental distribution of Anemone section Anemone (Ranun- culaceae). Botanical Journal of the Linnean Society 160, 312– 354. Endress, P. K., 1995: Floral structure and evolution in Ranuncula- ceae. Plant Systematics and Evolution 9 (Supplement), 47–61. Erbar, C., Kusma, S., Leins, P., 1999: Development and interpreta- tion of nectary organs in Ranunculaceae. Flora 194, 317–332. Erbar, C., Leins, P., 2013: Nectar production in the pollen fl ower of Anemone nemorosa in comparison with other Raunculace- ae and Magnolia (Magnoliaceae). Organism Diversity and Evolution 13, 287–300. Erdtman, G., 1954: An introduction to pollen analysis. Chronica Botanica Company. Faegri, K., Van Der Pijl, L., 1979: The principles of pollination ecology. 3rd edn., Pergamon Press, Oxford. Friedman, J., Barrett, S. C. H., 2009: Wind of change: new in- sights on the ecology and evolution of pollination and mating in wind-pollinated plants. Annals of Botany 103, 1515–1527. Gostin, I. N., 2009: Scanning electron microscopy investigations regarding Adonis vernalis L. fl ower morphology. Analele Universităţii din Oradea, Fascicula Biologie 16, 80–84. Hanley, M. E., Lamont, B. B., Fairbanks, M. M., Rafferty, C. M., 2007: Plant structural traits and their role in antiherbivore de- DENISOW B., ANTOŃ S., WRZESIEŃ M. 80 ACTA BOT. CROAT. 75 (1), 2016 fense. Perspectives in Plant Ecology, Evolution and Systemat- ics 8, 157–78. Hedhly, A., 2011: Sensitivity of fl owering plant gametophytes to temperature fl uctuations. Environmental and Experimental Botany 74, 9–16. Hegi, G., 1974: Illustrierte Flora von Mitteleuropa. Band III, Teil 3, Lieferung 2/3:203. Verlag P. Parey, Berlin, Hamburg. Horovitz, A., Galil, J., Zohary, D., 1975: Biological fl ora of Israel. 6. Anemone coronaria. Israel Journal of Botany 24, 26–41. Horovitz, A., 1991: The pollination syndrome of Anemone coro- naria L.; an insect-biased mutualism. Acta Horticulturae 288, 283–287. Karabourniotis, G., Papadopoulos, K., Papamarkou, M., Manetas, Y., 1992: Ultraviolet-B radiation absorbing capacity of leaf hairs. Physiologia Plantarum 86, 414–418. Keijzer, C. J., 1987: The process of anther dehiscence and pollen dispersal. 1. The opening mechanism of longitudinally dehisc- ing anthers. New Phytologist 105, 487–498. Konarska, A., 2014: Micromorphology and anatomy of fl owers and nectaries of Saxifraga stolonifera L. Acta Agrobotanica 67, 3–12. Kwiatkowska-Falińska, A., Faliński, J. B., 2007: Conditions of the occurrence of Anemone sylvestris in a Kettle Hole in North- Eastern Poland. Acta Societatis Botanicorum Poloniae 76, 133–140. Maciejewska-Rutkowska, I., Antkowiak, W., 2013: Taxonomic utility of achene morphology and anatomy in Anemone L. (Ranunculaceae) species. Acta Biologica Cracoviensia Series Botanica 55, 29–36. Müller, S., 2002: Diversity of management practices required to ensure conservation of rare and locally threatened plant spe- cies in grasslands: a case study at a regional scale (Lorraine, France). Biodiversity and Conservation 11, 1173–1184. Nishikawa, Y., Kudo, G., 1995: Relationship between fl ower num- ber and reproductive success of a spring ephemeral herb, Anemone fl accida (Ranunculaceae). Plant Species Biology 10, 111–118. Ordway, E., 1986: The phenology and pollination biology of Anemone patens (Ranunculaceae) in Western Minnesota. Pro- ceedings The prairie: past, present, and future: proceedings of the Ninth North American Prairie Conference, 31–34. Piękoś-Mirkowa, H., Mirek, Z., 2006: Rośliny chronione. Multico Ofi cyna Press, Warszawa, (in Polish). Roulston, T. H., Cane, J. H., 2000: Pollen nutritional content and digestibility for animals. Plant Systematics and Evolution 222, 187–209. Sulborska, A., Dmitruk, M., Konarska, K., Weryszko-Chmielews- ka E., 2014: Adaptation of Lamium album L. fl owers to polli- nation by Apoidea. Acta Scientiarum Polonorum. Hortorum Cultus 13, 31–43. Szklanowska, K., 1995: Pollen fl ows of crowfoot family (Ranun- culaceae L.) from some natural plant communities. In: Ban- aszak, J. (ed.), Changes in fauna of wild bees in Europe, 201– 209. Pedagogical Univ. Bydgoszcz (1st ed). Tamura, M., 1995: Angiospermae. Ordnung Ranunculales. Fam. Ranunculaceae. II. Systematic Part. In: Hiepko, P. (ed.), Natürliche Pfl anzenfamilien, 223–519. Second ed., Duncker & Humblot, Berlin, Germany. Waldbauer, G. P., Friedman, S., 1991: Self-selection of optimal di- ets by insects. Annual Review of Entomology 36, 43–63. Willmer, P., 2011: Pollination and fl oral ecology. Princeton Univ Press, Princeton. Wrzesień, M., Denisow, B., 2006: The share of nectariferous and polleniferous taxons in chosen patches of thermophilous grasslands of the Lublin Upland. Acta Agrobotanica 59, 213– 221. Van Doorn, W.G., Van Meeteren, U., 2003: Flower opening and clos- ure: a review. Journal of Experimental Botany 54, 1801–1812. Zając, M., Zając, A., 2009: The geographical elements of native fl ora of Poland. Laboratory of Computer Chorology, Institute of Botany, Jagiellonian University, Kraków. Ziman, S., Bulakh, E., Tsarenko, O., 2011: Anemone L. (Ranuncu- laceae): comparative morphology and taxonomy of the spe- cies from the Balkan fl ora. Botanica Serbica 35, 87–97.