- Email: [email protected]
Reversible anther opening enhances male fitness in a dichogamous aquatic plant Butomus umbellatus L., the flowering rush
Aquatic Botany 99 (2012) 27–33
Contents lists available at SciVerse ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Reversible anther opening enhances male fitness in a dichogamous aquatic plant Butomus umbellatus L., the flowering rush Jun Li a , Qing-Feng Wang b,∗ , Robert Wahiti Gituru c , Chun-Feng Yang b,∗ , You-Hao Guo a a b c
College of Life Sciences, Wuhan University, Wuhan 430072, China Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, The Chinese Academy of Sciences, Wuhan 430074, China Botany Department, Jomo Kenyatta University of Agriculture and Technology, 62000-00200 Nairobi, Kenya
a r t i c l e
i n f o
Article history: Received 10 August 2011 Received in revised form 17 January 2012 Accepted 24 January 2012 Available online 2 February 2012 Keywords: Outward bending of anther wall Pollen longevity Pollen viability Pollen/ovule ratio Pollination Reabsorption
a b s t r a c t We studied reversible anther opening in a dichogamous aquatic plant Butomus umbellatus L. (Butomaceae) to assess its consequence on male fitness. Light microscope observations indicated that stomium breakage was simultaneous in all the anthers within an opening flower; however, detachment of the epidermis and outward bending of the anther wall were asynchronous. SEM observations showed that epidermis cells alternated between orbicular and crinkly shapes in response to absorption and loss of water. This generated centripetal and centrifugal forces which were significant enough to cause inward and outward bending of the anther wall, thus causing opened anthers to close and closed anthers to re-open respectively, depending on relative humidity of the environment. Behaviour of in vitro and in situ anthers from different whorls within a flower was recorded under four regimes of relative humidity or under a water-spraying treatment. The three stages of anther opening namely stomium breakage, epidermis detachment and outward bending of the anther wall were affected differently by moisture levels. Outward bending of anther wall which was responsible for reversible anther opening was mainly dependent on environmental relative humidity levels. Notably, on sunny days, anther re-opening was inhibited at high relative humidity for in vitro anthers, but not for anthers in situ. Reabsorption, rather than evaporation was responsible for reversible anther opening for this aquatic plant. Water-spraying treatment indicated that flowers with re-closed anthers attracted fewer pollinators. Results using pollen stainability and germinability of pollen on stigmas for pollen grains from different treatments also showed the mechanism of reversible anther opening significantly prolonged pollen longevity by maintaining high viability in a simulated rainy. Discontinuous pollen presentation due to reversible anther opening avoided unfavorable pollination environment and pollen loss. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Modes of pollen dispersal and pollen longevity are expected to maximize male fitness of a flowering plant, and may involve adaptations to certain biotic (e.g. insects, bats, birds, etc.) or abiotic (e.g. wind, water) agents of pollination (Faegri and van der Pijl, 1979; Harder and Thomson, 1989; Bernhardt, 1996; Zhang et al., 2010). As a prerequisite of pollination, anther opening has been widely studied with regard to the mechanical and ecophysiological mechanisms of dehiscence (Buchmann, 1983; Keijzer, 1987; Pacini, 2000; Carrizo García et al., 2006). Selective anther opening may respond to pollination environment and enhance pollen dispersal and survival (Castellanos et al., 2006; Passarelli and Cocucci, 2006).
∗ Corresponding authors. Tel.: +86 27 87510526; fax: +86 27 87510526. E-mail addresses: [email protected] (Q.-F. Wang), [email protected] (C.-F. Yang). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2012.01.003
In general, anther opening is a result of tissue desiccation (Schmid, 1976; Buchmann, 1983; Keijzer, 1987). However, it is noteworthy that the dehiscence of the anther can be caused by either evaporation (Keijzer, 1987; Bonner and Dickinson, 1990; Keijzer et al., 1996; Matsui et al., 1999) or reabsorption (Keijzer et al., 1987; Carrizo García et al., 2006). Anther opening due to evaporation or reabsorption must have different responses to relative humidity (RH) and this may potentially affect adaptation of the pollination system. Anther dehiscence due to evaporation must rely to a greater degree on environmental factors while that due to reabsorption is likely to be more dependent on the plant’s own physiological response to environmental change. For example, at high RH, reabsorption, rather than evaporation may occur and result in anther opening. Adaptive linkages between anther characteristics and pollination system have been reported in some taxa, such as Penstemon (Castellanos et al., 2006) and Solanum (Carrizo García and Barboza, 2006; Passarelli and Cocucci, 2006). Antherwall structure affecting pattern of pollen release was found to be correlated with pollination syndrome.
28
J. Li et al. / Aquatic Botany 99 (2012) 27–33
Fig. 1. Characteristics of inflorescence, flower and anther for B. umbellatus. (A) Sequential blooming of flowers (with long pedicel) within an inflorescence (with long stalk); (B–E) asynchronous dehiscence of anthers within a flower: (B) nine undehisced anthers within a flower; (C) early dehiscence of anthers of the outer whorl; (D) anthers of the inner whorl dehiscence later; (E) dehiscence of all the anthers within a flower; stigmas disjoined and becoming receptive; (F) anthers re-closing after water-spraying treatment. Scale bars: 1 cm.
In this study we found that for the aquatic plant Butomus umbellatus L. (the flowering rush) anther opening via longitudinal dehiscence can be reversed when the anther is wetted as happens during rainy weather. Additionally, the flower is dichogamous and the duration of the male phase is quite short. To ascertain the structural and ecophysiological mechanisms of this novel bidirectional process and its possible adaptive significance, experiments were conducted to investigate the following: (1) structural changes of the anther wall during opening and closing; (2) the pattern of environmental factors affecting anther opening and closing both for an individual anther and for the anthers within a flower; (3) change in pollen viability during anthesis and the effect of anther re-closing on pollen viability; and (4) the link between pollinator and anther behaviour.
Pollination observations were conducted in natural populations over 120 h in July 2006, July to August 2007 and July 2009 in which we recorded pollinator type and frequency for each individual plant during rainy episodes, during the intervals (longer than 30 min) between the rainy episodes, as well as during sunny periods. Pollination frequency was estimated as the cumulative period of time spent by pollinators on an umbel in a 1-h observation period. Ten randomly-selected umbels were used during the peak blooming period to calculate the average pollination frequency. Pollinators were also captured during their visits to the flowers and taken to the laboratory for identification. To check for potential autogamy or apomixis, 20 bagged flowers were marked and their fruits later harvested to assess seed set. 2.2. Factors influencing anther behaviour
2. Materials and methods 2.1. Study species B. umbellatus is an emergent, aquatic monocot in a monotypic family that inhabits shallow water around the margins of lakes and slow-moving rivers. Inflorescences are cymose umbels borne on thin, cylindrical, upright stalks that consist of 15–50 pink flowers (Fig. 1A). Flowers contain nectaries and each consists of six conduplicate carpels. The androecium of each flower consists of nine stamens (in an outer whorl of six and an inner whorl of three) (Sun, 1992; Bhardwaj and Eckert, 2001; Fig. 1). Our study was conducted on a natural population at the Wild-Lotus-Garden (45◦ 30 N, 131◦ 51 E), Mishan county, Heilongjiang Province, China during July 2006, July to August 2007, and July 2009. Fifty plants were randomly selected and enclosed with fine bridal veil netting to exclude pollinators. The plants were used to estimate the seed production in the absence of pollinators as well as to provide pollen grains for experiments of pollen stainability since pollen removal by pollinators on fine weather days was quite rapid in the study populations.
Opening flowers were randomly selected at 0800 h before anthers dehisced for observation of anther behaviour. We first recorded the sequence and duration of anther opening within a flower. To observe the detailed behaviour of anthers under conditions of wetness, individual flowers with dehisced anthers were sprayed with rainwater. An atomizer was used to spray each individual flower during a sunny day to simulate rain. The anthers were then photographed to record changes in their structure during water loss on the flower. To investigate the relationship between corolla status (wet/dry) and anther behaviour, we recorded the proportion of closed/re-opened anthers out of the total number of anthers which had dehisced for flowers with dry or wet corollas respectively during sunny periods. Additionally, to evaluate the correlation between corolla status and pollinator visitation, we recorded the percentage of visited flowers out of the total number of flowers with dry and wet corollas respectively during sunny periods. In order to better understand the environmental cues that control the opening and re-closing of anthers, at least 30 freshly opened flower buds (randomly selected on ten individual plants) were
J. Li et al. / Aquatic Botany 99 (2012) 27–33
subjected to variable relative air humidity (RH), low temperature and low light intensity (darkness), respectively. The condition of low temperature was simulated using a transparent plastic bucket containing ice while low light intensity was simulated by fitting a bucket with a cover. Four levels of RH (20%, 50–55%, 70–75%, and 95%) were used in this study. Two levels of RH were created following the methods of Winston and Batesm (1960) using saturated salt solutions in a box covered with transparent plastic film (20% RH with potassium acetate and 95% RH with potassium dichromate). The other two RHs (50–55%, 70–75%) represent the normal ranges of variation in RH in the laboratory and outdoors at the lakeside, respectively at the study site from 0830 to 1700 h on a sunny day. Anther behaviour was observed under the four levels of RH from opened flower buds which were picked at 0800 h and placed in water in vials covered with plastic film. The pedicles were inserted through small holes in the plastic film. Vials with flower buds were placed under the different conditions for observation of anther behaviour. Further in anticipation of potential differences in anther behaviour under conditions of high RH (as occur on rainy days) between anthers in situ and those in vitro, two plants were temporarily enclosed with transparent plastic film to create the condition of high RH (90–95%). Flowers on the two plants were marked at 0800 h before the onset of anther dehiscence and used for observing anther behaviour. 2.3. Structural aspects of anther behaviour To investigate the structural changes of the anther wall during anther opening and re-closing, flower buds and anthers in various states of opening (closed, opened, intermediate, and reclosed) from natural populations were collected and immediately fixed in FAA solution (formalin:acetic acid:70% ethanol at a ratio of 5:6:89 by volume) for laboratory analyses. The surfaces of the anther epidermis (outer wall) were observed by scanning electron microscopy (SEM). Fixed anthers for SEM were subjected to a dehydration series to 100% ethanol, and then dried in CO2 to the critical point, coated with gold, and observed using a Hitachi S-450 SEM at 20 kV. Anthers in different stages were also used to make sections for observation under light microscope. The fixed anthers were dehydrated in a graded ethanol series and then left overnight in 100% ethanol. The material was then passed through graded ethanol/xylene mixtures (100% ethanol; 1:1; and 100% xylene) and embedded through graded xylene/paraffin mixtures (1:1; 100% paraffin). Serial sections (2 m) were obtained with a microtome and stretched on glass slides, then exposed to xylene–ethanol series to remove paraffin and stained with 0.1% safranin.
29
percentage of stained pollen grains out of the total number of pollen grains on a slide. The average for the three samples was regarded as the pollen stainability of an individual flower. To measure the duration of pollen viability, pollen stainability was checked at 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and 36 h after anther dehiscence. To compare pollen viability from exposed and re-closed anthers, dehisced anthers from ten flowers (each from separate bagged plants) were maintained in a re-closed status until 7, 8, 12 and 24 h after anther opening. To maintain anthers in the re-closed status, flowers were treated to intermittent water spraying using an atomizer. Pollen grains for estimation of pollen stainability were obtained from the anthers when they re-opened. To assess the effect of wetting on pollen stainability (and hence viability), fresh pollen grains from 20 flowers selected at random from ten individuals were dipped in water for 30 min and then stained. Since a correlation between fluorochromatic reaction or germinability in vitro and overall pollen competence may be absent (Thomson et al., 1994), we estimated the ability of pollen to germinate on fresh stigmas for pollen grains under different treatments in comparison with control group. Pollen grains from exposed and enclosed anthers on the same plant were applied to different fresh stigmas within a flower 12 and 24 h after anther opening, respectively. To do this, individual stigmas from enclosed flowers at the beginning of the female phase was numbered sequentially and hand-pollinated with the different pollen grains using a brush. Hand pollination was conducted using fresh pollen grains as well as using pollen grains from the same flower but which had been dipped in water. Ten hours after pollination, stigmas with different pollen load were collected and fixed immediately in FAA solution. To estimate pollen performance on stigma, stigmas were observed under a fluorescence microscope (Nikon 80i) after treatment with 8 mol L−1 NaOH for 12 h, followed by dyeing in 0.1% aniline blue. For each treatment, at least 30 pistils were used to estimate pollen performance on the stigmas. A stigma with successful pollen germination was defined as one that recorded 10 or more germinated pollen grains. For pollen and ovule counts, 20 open flowers with no dehisced anthers from 20 randomly selected plants were fixed in FAA solution for laboratory examination following the methods of Yang and Guo (2004). The pollen–ovule ratio was calculated by dividing the total number of pollen grains (nine anthers) by the total number of ovules produced in a flower (six pistils). Data in the study are shown as mean ± S.D. One-way ANOVA was used to compare the changes of pollen stainability under different treatments.
2.4. Pollen viability and pollen–ovule ratio
3. Results
Pollen stainability is commonly used as an indicator of pollen viability (Dafni and Firmage, 2000). In this study we considered stainability of pollen with 2,5-diphenyl tetrazolium bromide (MTT) as an indication of pollen viability. MTT detects the presence of dehydrogenase in viable pollen (Rodriguez-Riano and Dafni, 2000; Zhang et al., 2010). To assess the ability of the technique to discriminate between viable and non-viable pollen, we also used the dye on heat-treated (boiled in water at 100 ◦ C for 2 h) pollen. In the present study, pollen stainability was used for estimating three aspects of pollen viability viz. duration of viability, and differences in viability of pollen from exposed and re-closed anthers and when dipped into water or not. For each treatment, pollen grains from ten flowers on bagged individuals were used. Pollen grains were collected from all anthers within a flower and randomly deposited on three slides (at least 1000 pollen grains per slide) using a fine brush for pollen staining. Estimation of pollen stainability followed the methods of Zhang et al. (2010). Pollen stainability was calculated as the
3.1. Floral biology Sexual reproduction in the flowering rush is dependent on pollen transfer by pollinators. Flowers which were excluded from pollinators set no seed. Eristalis species were the most frequent pollinators. In general, the release of pollen grains relied solely on mechanical contact with the pollinator rather than on shaking of the anther or on gravity. Results showed that pollinator frequency was high (21.6 ± 3.5, N = 36) on sunny days, moderately high (4.2 ± 2.1, N = 28) during the intervals between the rainy episodes (intervals longer than 30 min), and close to zero during rainy episodes. Results of pollinator visitation following the waterspraying treatment showed that corolla wetting clearly affected pollinator behaviour. Flowers with dry corollas received more visitation (90%, N = 96) than those with wet corollas (8%, N = 78). In fine weather, an anther dispersed its entire pollen grains within 3 h of opening while the entire pollen grains in a flower
30
J. Li et al. / Aquatic Botany 99 (2012) 27–33
were removed within 12 h. Under natural conditions, the six aggregate pistils separate at about 12 h after the end of the male function. Stigmas then expand and become receptive sequentially within 6 h. The ivory-colored stigmas become dry and lackluster after 5–7 h by which time they have received pollen grains (Fig. 1A and E). Under natural conditions flower longevity of the species in our study area is about 72 h. 3.2. Factors influencing anther behaviour Within a flower, the anthers presented pollen grains sequentially from 0800 to 1830 h (Fig. 1B–E). No stomata occur on the anther and fresh anthers do not open at night. During anthesis, the six anthers in the outer whorl exposed pollen grains earlier than the three anthers in the inner whorl (Fig. 1C and D). Generally, the average interval between opening of the last anther of the outer whorl and the first anther of the inner whorl is slightly longer than that between the opening of the first and the last anthers within a whorl (see also Fig. 1D). Breakage of the stomium of endothecium in all the anthers within a flower was complete by the time the flower opened. Sequential opening of individual anthers was due to variation among anthers in time for rupture of epidermal cells. Endotheciums of all 63 anthers from opening flower buds were broken. Epidermis cells at the front of stomium in 47 (74.6%) of the anthers were intact while those in the other 16 (25.4%) anthers were ruptured in readiness for the outward bending of the anther wall. No difference was detected in the ability of the anther to re-close between the anthers of the inner and those of the outer whorls in a flower (Fig. 1F). At 20% RH, anther opening (thus exposing pollen grains) was slightly accelerated for the inner whorls but not for the outer whorls. At this level of RH all anthers within a flower had presented their pollen grains before 1500 h. The same pattern was found at 50–55% RH. However, at 95% RH, the time for exposure of pollen grains in both whorls was delayed. At this level of RH approximately 16.0% of the anthers in the outer whorl and approx. 42.2% of the anthers in the inner whorl had no exposed pollen grains by 2000 h. However, for anthers in situ at 90–95% RH, nearly all anthers had presented pollen grains before 1900 h. Furthermore, for unopened in vitro anthers at this RH level the epidermis cells of the stomium had split before 1700 h. When the covering was removed causing RH to drop to 70–75% (environmental RH), the anthers opened within 30 min. Experiments indicated that a one-time treatment of low temperature, high humidity or low light intensity (darkness) did not cause dehisced anthers to re-close. When low temperature and high humidity treatments were combined the resultant condensation created water drops which caused the wetted anthers to re-close. In the simulated rain (spraying) treatment opened anthers re-closed within 4–7 min (5.4 ± 0.8; Fig. 1F). The proportion of re-opened anthers was significantly higher for dry corollas (85%, N = 584) than for wet corollas (11%, N = 389). Closed anthers re-opened within 13–20 min (16.3 ± 2.5) after the corollas dried. The time taken for closed anthers to re-open increased with increase in RH until at 95% RH (for anthers in vitro) it became very infrequent for closed anthers to re-open. 3.3. Structural aspects of anther behaviour Anther opening in B. umbellatus occurs by a longitudinal slit in the side of the anther. The anther wall consists of an epidermis and an endothecium. The later structure has cells with U-shaped wall thickenings (Fig. 2A–C). However, the cells of endothecium in the septa in both locules lack these wall thickenings (Fig. 2A). Unlike the rest of the epidermal cells which enlarged considerably, the epidermal cells facing the septum remained small (Fig. 2A). The breakage
of endothecium in the stomium started during shrinking of the septum. This was followed by opening of the anther after detachment of the adjacent epidermis cells on both sides of the stomium. Once the anther was opened along its entire length, the walls started to shrink and bend outwards. The ventral walls bend until they touch each other, while the dorsal walls bend only partially forming a concave surface. Anther length was noticeably shortened by the outward bending (Fig. 1C and D). The process of anther opening starting from rupture of the epidermis cells to the complete bending outwards of the anther wall could be reversed when the anthers were wetted by spraying with water or by rainfall. Photographs under SEM indicated that the retractile cells of epidermis alternated from the orbicular to the crinkly status when anthers re-closed and re-opened respectively (Fig. 2D and E). Results indicated that epidermis cells were flattened in tangential view and the cells of endothecium were stacked in transverse view when anthers were closed (Fig. 2B). As the anthers opened (re-opened), epidermis cell were compact and their tangential length was noticeably shortened while the cell of endothecium were crinkled and their transverse length was noticeably reduced (Fig. 2C). 3.4. Pollen viability and pollen–ovule ratio Pollen of the species is monosulcate and coated with very little pollenkitt. Results of MTT staining indicated that pollen grains maintained the highest pollen viability during the first 7 h, and then gradually lost viability until they were no longer viable 24 h after anther dehiscence (Fig. 3). Viability decreased rapidly within 30 min in pollen which had been dipped in water (from 57% ± 7% to 14% ± 6%, respectively). However, in the water-spraying experiment pollen grains in re-closed anthers maintained high viability for 12 h which decreased to moderately high viability by 24 h after anther dehiscence (Fig. 3). Moreover, for pollen grains which had dipped in water for 30-min or exposed for 12 h or longer on a dehisced anther, it was difficult to detect any pollen germination on stigma. For stigmas with exposed pollen grains, one out of 31 stigmas (3%) was found to have successful pollen germination after 12 h of exposure. No germination was detected after 24 h of exposure. Results also indicated that successful pollen germination on stigma following pollination with pollen which had been dipped in water for 30-min was low (21%). In contrast, successful pollen germination was found on all stigmas with fresh pollen load. However, the proportion of stigmas with successful pollen germination for enclosed pollen grains from re-closed anthers after 12 and 24 h was relatively high (87% at 12 h and 53% at 24 h respectively). The nine anthers and six pistils of a single flower of B. umbellatus produce on average 4.26 × 105 pollen grains (N = 20, S.D. = 0.54 × 105 ), and 918.1 ovules (N = 20, S.D. = 247.2) respectively. The pollen–ovule ratio in the species was 463.9. 4. Discussion To accomplish pollination, aquatic plants have evolved special mechanisms adapted to water environment (Cox, 1988). For some species of Hydrocharitaceae with hydrophilous pollination, including Vallisneria spiralis and Enhalus acoroides, anthers or flowers carry dry pollen across the water surface to receptive stigma. For the entomophilous B. umbellatus, the unique mechanism of reversible anther opening is of adaptive meaningfulness. Anther behaviour in the species is adapted to the pollination environment and thus enhances the male fitness. 4.1. Anther behaviour Although the mechanism of anther dehiscence and the physiological processes involved in the process are different in different
J. Li et al. / Aquatic Botany 99 (2012) 27–33
31
Fig. 2. Structural aspects of anther in different status. (A–C) Cross sections (sides of epidermis and endothecium are indicated by E and H, respectively): (A) mature undehisced anther without dehiscence; note U-shaped thickenings in cells of endothecium (blue arrow), no U-shaped thickenings in cells of septum (short black arrow), and small epidermis cells at the front of septum (to forming stomium, long black arrow); (B) closing (re-closing) anther wall; note flattened epidermis cells in tangential view and columniform cells of endothecium in transverse view (blue arrow indicating U-shaped thickenings in cells of endothecium); (C) opening (re-opening) anther wall; note compact epidermis cells and squeezed cells of endothecium; (D and E) SEM photograph of epidermis: (D) closing (re-closing) anther; note orbicular epidermis cells; (E) opening (re-opening) anther; note crinkly epidermis cells. Scale bars: A, 100 m; B and C, 20 m; and D and E, 10 m.
species, studies have shown that the structural basis of anther dehiscence in longitudinally dehiscing anthers follows the process summarized by Keijzer (1987) and reported by Carrizo García et al. (2006) as well as in our present study. The finding that anthers in freshly opening flower buds already had endothecium of the their stomium broken but that they were separated in time for epidermis detachment and the outward bending of anther wall is an important finding demonstrating the
asynchronous pattern in this natural process. The results indicated that the three sequential steps of anther opening may be regulated differently in relation water control (see also Carrizo García et al., 2006 for Allium triquetrum). First, stomium breakage is likely a developmentally programmed process related to the development of wall thickenings (Mitsuda et al., 2005). Following the formation of the wall thickenings stomium breakage appeared to be independent of environmental RH. Second, epidermis detachment must be
Fig. 3. Pollen viability of B. umbellatus indicated by MTT staining between exposed pollen grains and pollen grains in closed anthers. Sites with different letter are significantly different at level P < 0.001.
32
J. Li et al. / Aquatic Botany 99 (2012) 27–33
under the control of the plant’s internal physiological mechanisms as evidenced by the observation that the epidermis cells of all the anthers detached once a particular time was reached irrespective of the RH values. Third, unlike the other two stages of anther opening, the outward bending of the anther wall is mainly controlled by moisture levels since higher or lower RH values inhibit or facilitate the process. As may be expected, the changes in anther behaviour in response to environmental moisture levels were more likely to occur at this stage. The mechanism and process of reversible anther opening has been rarely studied since anther opening was always regarded as a unidirectional process (but see Edwards and Jordan, 1992; Wang et al., 2009). In B. umbellatus, this bidirectional process might be ascribed mainly to the retractile epidermis cells. We hypothesize that when the anther is dehydrated, the cells of the epidermis become crinkly and squeeze the endothecium cells, which results in shortening of the epidermis cells and a reduction in the transverse length of the endothecium cells. Consequently, an outward centrifugal force is generated which causes bending of the anther wall. Conversely when the anther rehydrated, a centripetal force arising from the returning of the epidermis and endothecium cells to their pre-dehydration state causes the anther wall to bend inwards (see also Vasil, 1967; Gerenday and French, 1988). Anthers in B. umbellatus opened (re-opened) at different levels of RH. This may indicate that evaporation and reabsorption operate in concert. Evaporation leads to low levels of RH accelerating anther opening (re-opening); however, the opening (re-opening) of the anthers which occurs at high RH suggests that reabsorption must play a role in the processes. Reabsorption may happen more easily for species with long filaments, pedicels, or inflorescence stalks in which water can be transferred (see also Keijzer et al., 1987; Carrizo García et al., 2006). For B. umbellatus, the conspicuously long pedicels and inflorescence stalks (Fig. 1A) may enhance reabsorption. Reabsorption rather than evaporation regulating anther opening is of greater adaptive significance for aquatic plants since RH in the natural habitats of these plants is commonly high. Evaporation has always been regarded as the main cause of the outward bending of anther wall in anther opening (Keijzer et al., 1996; Carrizo García et al., 2006). In the present study, the observation that re-closed anthers in vitro did not re-open even at high RH levels but that in situ the anthers re-opened at 95% RH may indicate that reabsorption rather than evaporation regulated the outward bending of anther wall. In view of the key role of reabsorption in reversible anther opening for aquatic plants, we hypothesize that epidermis cells are hydrophilic and at high RH they absorbed water readily which resulted in re-closing of the anther. Since the anthers have no stoma the water in them is transferred to tissues of adjacent structures (pedicel and stalk) when these tissues were not saturated. This caused the anthers to re-open. This may provide an explanation as to why anthers did not open (re-open) during the night and in rainy weather, but they did so under conditions of high RH. During night time it was difficult to transfer the water in the anther to adjacent tissues owing to the absence of photosynthesis (hence low concentration of solutes) in the plant. Similarly the saturated status of the tissues of the pedicel and stalk during rainy weather hampered transfer of water from the anthers and thus prevented the anthers from opening (re-opening). Under the water-spraying treatment, anthers of flowers with wet corollas did not open (re-open) possibly due to the saturated status of adjacent tissues which hampered transfer of water from the anthers. 4.2. Reversible anther opening enhances male fitness Reversible anther opening has been reported in very few species and is limited to taxa with extended flower longevity such as Lilium philadelphicum (longer than 9 d) (Edwards and Jordan, 1992) and
Paris polyphylla var. yunnanensis (longer than 20 d) (Wang et al., 2009). From the viewpoint of adaptation, species with short-lived flowers or high pollination efficiency do not require to evolve the mechanism of reversible anther opening (see also Edwards and Jordan, 1992). For the dichogamous species B. umbellatus, the duration of male phase was quite short and the efficiency of pollen removal was relatively high owing to the exposed pollen presentation and high pollination frequency (see also Bhardwaj and Eckert, 2001). The unexpected finding of reversible anther opening in this species enriched our understanding of evolution of anther biology. Clearly, the significance of reversible anther opening was enhancement of male fitness by prolonging male duration or avoiding unfavorable environment. Reversible anther opening must achieve this in two ways: Firstly, re-closing anthers help to retain pollen viability since pollen grains in these anthers must take a longer time before they are deposited on stigmas. Secondly, pollinator behaviour must be matched with anther behaviour to ensure relatively rapid pollen export. B. umbellatus pollen may be categorized as partially dehydrated, orthodox type pollen according to its desiccation tolerance (longer than 6 h) and monosulcate shape (Nepi et al., 2001, 2010; Zhang et al., 2010; Franchi et al., 2002, 2011). In the present study, results of pollen staining with MTT indicated that anther re-closing caused pollen to remain viable for a relatively longer period. The maintenance of pollen viability in re-closed anthers might be due to the fact that the re-closed anther created a microenvironment with favorable RH and temperature (see also Stanley and Linskens, 1974; Faegri and van der Pijl, 1979). In addition, the re-closing of the anther avoided waste of pollen grains because pollen grains on an exposed anther may be easily washed away under heavy rain. The phenomenon ensured rapid change of anther behaviour in response to fluctuating weather conditions ensuring the quality of pollen export and prolonging the duration of male phase. In the study area the flowers of B. umbellatus attracted eight insects. The exposed pollen grains are clump in shape due to stickiness and easily adhered to insects visiting the flowers (see also Fernando and Cass, 1997). The efficiency of pollen export for the species was quite high (see also Bhardwaj and Eckert, 2001). However, even at high level of RH during interval of showers, high records of instances of pollinator visitation were made because of the broad pollinator fauna in the species (see also Fernando and Cass, 1997). Correspondingly, re-closed anthers would re-open when the interval between two rainy episodes was longer than 30 min. Results rain simulation (spraying) treatment on sunny days indicated that flowers with wet corolla did not re-open their anthers and they also attracted no pollinators. As evaporation (reabsorption) of water on the corolla occurred, pollinators visited the flowers and the re-closed anthers on the flowers re-opened. According to the criterion of Cruden (1977), the breeding system of B. umbellatus falls into the category between facultative autogamy (168.5) and facultative xenogamy (796.6) with a pollen–ovule ratio of 463.9. Clearly, the dichogamous species B. umbellatus belongs to category of xenogamous taxa. In comparison to other xenogamous species, the low pollen–ovule ratio indicates higher male fitness in the species (Queller, 1984). However, Fernando and Cass (1997) suggested that the developmental irregularities such as meiotic aberration, pollen abortion, excessive winding of pollen tubes and rapid decrease of pollen viability might reduce male reproductive potential. There should be compensatory mechanism enhancing male potential because the sequential anthesis within an inflorescence and staggered anther dehiscence within a flower may increase the chances of pollen to be transferred (Fernando and Cass, 1997). Furthermore, plants of B. umbellatus flowered during the rainy season in Northeastern China (summer rainfall accounted for 90% of the annual total). The remarkable mechanism of reversible anther opening confers on the plant adaptation to the frequent
J. Li et al. / Aquatic Botany 99 (2012) 27–33
scattered showers common at this time of year. Discontinuous pollen presentation effectively prolonged the duration of male phase and avoided unfavorable pollination environment. The unique anther behaviour observed in B. umbellatus might, to a certain extent, by way of enhancing male fitness responsible for the disproportional relationship between pollen–ovule ratio and breeding system in the species (see also Armstrong, 1992; Edwards and Jordan, 1992; Han et al., 2008). The mechanism of reversible anther opening in response to rain may be ascribed mainly to the retractile epidermis cells of anther wall. Reabsorption clearly plays important role in anther opening and re-opening in this aquatic plant with long pedicle and inflorescence stalk. This novel mechanism significantly prolonged pollen longevity by maintaining high pollen viability. The evolving of reversible anther opening in this dichogamous plant with a relatively short flower span is a rather unexpected occurrence. Anther itself controlling pollen dispersal and creating discontinuous pollen presentation avoided unfavorable pollination environment and pollen loss and enhanced pollen competence, thus making the plant better adapted to pluvious flowering season and enhancing male fitness. Acknowledgements We thank Jan Vermaat and two anonymous reviewers for their helpful suggestions and comments, Jin-Ming Chen and Zhi-Yuan Du for assisting in fieldwork, Zhi-Xin Zhu for preparation of the paraffin sections, Xiao-Lin Zhang for MTT staining, Qian Yu for valuable discussions, and Hong Liu (Institute of Zoology, CAS) for assisting in insect identification. This work was supported by the National Natural Science Foundation of China (Grants 31070206 to C.-F.Y and 31170214 to Y.-H.G). References Armstrong, J.E., 1992. Lever action anthers and the forcible shedding of pollen in Torenia (Scrophulariaceae). Am. J. Bot. 79, 34–40. Bernhardt, P., 1996. Anther adaptations for animal pollination. In: D’Arcy, W., Keating, R. (Eds.), The Anthers: Form, Function and Phylogeny. Cambridge University Press, New York, pp. 192–220. Bhardwaj, M., Eckert, C.G., 2001. Functional analysis of synchronous dichogamy in flowering rush, Butomus umbellatus (Butomaceae). Am. J. Bot. 88, 2204–2213. Bonner, J.L., Dickinson, H.G., 1990. Anther dehiscence in Lycopersicon esculentum Mill. II. Water relations. New Phytol. 115, 367–375. Buchmann, S.L., 1983. Buzz pollination in angiosperms. In: Jones, C.E., Little, R.J. (Eds.), Handbook of Experimental Pollination. Van Nostrand Reinhold, New York, pp. 73–113. Carrizo García, C., Barboza, G.E., 2006. Anther wall development and structure in wild tomatoes (Solanum sect. Lycopersicon): functional inferences. Aust. J. Bot. 54, 83–89. Carrizo García, C., Nepi, M., Pacini, E., 2006. Structural aspects and ecophysiology of anther opening in Allium triquetrum. Ann. Bot. 97, 521–527. Castellanos, M.C., Wilson, P., Keller, S.J., Wolfe, A.D., Thomson, J.D., 2006. Anther evolution: pollen presentation strategies when pollinators differ. Am. Nat. 167, 288–296. Cox, P.A., 1988. Hygrophilous pollination. Annu. Rev. Ecol. Syst. 19, 261–280. Cruden, R.W., 1977. Pollen–ovule ratios: a conservative indicator of breeding systems in flowering plants. Evolution 31, 32–46.
33
Dafni., A., Firmage, D., 2000. Pollen viability and longevity: practical, ecological and evolutionary implications. Plant Syst. Evol. 222, 113–132. Edwards, J., Jordan, J.R., 1992. Reversible anther opening in Lilium philadelphicum (Liliaceae): a possible means of enhancing male fitness. Am. J. Bot. 79, 144–148. Faegri, K., van der Pijl, 1979. The Principles of Pollination Ecology, 3rd ed. Pergamon Press, Oxford. Fernando, D., Cass, D.D., 1997. Developmental assessment of sexual reproduction in Butomus umbellatus (Butomaceae): male reproductive component. Ann. Bot. 80, 449–456. Franchi, G.G., Nepi, M., Dafni, A., Pacini, E., 2002. Partially hydrated pollen: taxonomic distribution, ecological and evolutionary significance. Plant Syst. Evol. 234, 211–227. Franchi, G.G., Piotto, B., Nepi, M., Baskin, C.C., Baskin, J.M., Pacini, E., 2011. Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival. J. Exp. Bot. 62, 5267–5281. Gerenday, A., French, J.C., 1988. Endothecial thickenings in anthers of porate monocotyledons. Am. J. Bot. 75, 22–25. Han, Y., Dai, C., Yang, C.-F., Wang, Q.-F., Motley, T.J., 2008. Anther appendages of Incarvillea trigger a pollen-dispensing mechanism. Ann. Bot. 102, 473–479. Harder, L.D., Thomson, J.D., 1989. Evolutionary options for maximizing pollen dispersal of animal pollinated plants. Am. Nat. 133, 323–344. Keijzer, C.J., 1987. The processes of anther dehiscence and pollen dispersal. I. The opening mechanism of longitudinally dehiscing anthers. New Phytol. 105, 487–498. Keijzer, C.J., Hoek, I.H.S., Willemse, M.T.M., 1987. The processes of anther dehiscence and pollen dispersal. III. The dehydration of the filament tip and the anther in three monocotyledonous species. New Phytol. 106, 281–287. Keijzer, C.J., Leferink-ten Klooster, H.B., Reinders, M.C., 1996. The mechanics of the grass flower: anther dehiscence and pollen shedding in maize. Ann. Bot. 78, 15–21. Matsui, T., Omasa, K., Horie, T., 1999. Mechanism of anther dehiscence in rice (Oryza sativa L.). Ann. Bot. 84, 501–506. Mitsuda, N., Seki, M., Shinozaki, K., Ohme-Tagaki, M., 2005. The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17, 2993–3006. Nepi, M., Cresti, L., Guarnieri, M., Pacini, E., 2010. Effect of relative humidity on water content, viability and carbohydrate profile of Petunia hybrida and Cucurbita pepo pollen. Plant Syst. Evol. 284, 57–64. Nepi, M., Franchi, G.G., Pacini, E., 2001. Pollen hydration status at dispersal: cytophysiological features and strategies. Protoplasma 216, 171–180. Pacini, E., 2000. From anther and pollen ripening to pollen presentation. Plant Syst. Evol. 222, 19–43. Passarelli, L., Cocucci, A., 2006. Dynamics of pollen release in relation to anther-wall structure among species of Solanum (Solanaceae). Aust. J. Bot. 54, 765–771. Queller, D.C., 1984. Pollen–ovule ratios and hermaphrodite sexual allocation strategies. Evolution 38, 1148–1151. Rodriguez-Riano, T., Dafni, A., 2000. A new procedure to assess pollen viability. Sex. Plant Reprod. 12, 241–244. Schmid, R., 1976. Filament histology and anther dehiscence. Bot. J. Linn. Soc. 73, 303–315. Stanley, R.G., Linskens, H.F., 1974. Pollen: Biology, Biochemistry, Management. Springer-Verlag, Berlin. Sun, X.Z., 1992. Flora of China, vol. 8. Science Press, Beijing. Thomson, J.D., Rigney, L.P., Karoly, K.M., Thomson, B.A., 1994. Pollen viability, vigor, and competitive ability in Erythronium grandiflorun (Liliaceae). Am. J. Bot. 81, 1257–1266. Vasil, I.K., 1967. Physiology and cytology of anther development. Biol. Rev. 42, 327–373. Wang, D.K., Sun, G.F., Wang, L.F., Zhai, S.H., Cen, X.J., 2009. A novel mechanism controls anther opening and closing in Paris polyphylla var. yunnanensis. Chin. Sci. Bull. 54, 244–248. Winston, P.W., Batesm, D.H., 1960. Saturated solutions for the control of humidity in biological research. Ecology 41, 232–237. Yang, C.-F., Guo, Y.-H., 2004. Pollen size-number trade-off and pollen–pistil relationships in Pedicularis (Orobanchaceae). Plant Syst. Evol. 247, 177–185. Zhang, X.L., Gituru, R.W., Yang, C.F., Guo, Y.H., 2010. Exposure to water increased pollen longevity of pondweed (Potamogeton spp.) indicates different mechanisms ensuring pollination success of angiosperms in aquatic habitat. Evol. Ecol. 24, 939–953.
Contents lists available at SciVerse ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Reversible anther opening enhances male fitness in a dichogamous aquatic plant Butomus umbellatus L., the flowering rush Jun Li a , Qing-Feng Wang b,∗ , Robert Wahiti Gituru c , Chun-Feng Yang b,∗ , You-Hao Guo a a b c
College of Life Sciences, Wuhan University, Wuhan 430072, China Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, The Chinese Academy of Sciences, Wuhan 430074, China Botany Department, Jomo Kenyatta University of Agriculture and Technology, 62000-00200 Nairobi, Kenya
a r t i c l e
i n f o
Article history: Received 10 August 2011 Received in revised form 17 January 2012 Accepted 24 January 2012 Available online 2 February 2012 Keywords: Outward bending of anther wall Pollen longevity Pollen viability Pollen/ovule ratio Pollination Reabsorption
a b s t r a c t We studied reversible anther opening in a dichogamous aquatic plant Butomus umbellatus L. (Butomaceae) to assess its consequence on male fitness. Light microscope observations indicated that stomium breakage was simultaneous in all the anthers within an opening flower; however, detachment of the epidermis and outward bending of the anther wall were asynchronous. SEM observations showed that epidermis cells alternated between orbicular and crinkly shapes in response to absorption and loss of water. This generated centripetal and centrifugal forces which were significant enough to cause inward and outward bending of the anther wall, thus causing opened anthers to close and closed anthers to re-open respectively, depending on relative humidity of the environment. Behaviour of in vitro and in situ anthers from different whorls within a flower was recorded under four regimes of relative humidity or under a water-spraying treatment. The three stages of anther opening namely stomium breakage, epidermis detachment and outward bending of the anther wall were affected differently by moisture levels. Outward bending of anther wall which was responsible for reversible anther opening was mainly dependent on environmental relative humidity levels. Notably, on sunny days, anther re-opening was inhibited at high relative humidity for in vitro anthers, but not for anthers in situ. Reabsorption, rather than evaporation was responsible for reversible anther opening for this aquatic plant. Water-spraying treatment indicated that flowers with re-closed anthers attracted fewer pollinators. Results using pollen stainability and germinability of pollen on stigmas for pollen grains from different treatments also showed the mechanism of reversible anther opening significantly prolonged pollen longevity by maintaining high viability in a simulated rainy. Discontinuous pollen presentation due to reversible anther opening avoided unfavorable pollination environment and pollen loss. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Modes of pollen dispersal and pollen longevity are expected to maximize male fitness of a flowering plant, and may involve adaptations to certain biotic (e.g. insects, bats, birds, etc.) or abiotic (e.g. wind, water) agents of pollination (Faegri and van der Pijl, 1979; Harder and Thomson, 1989; Bernhardt, 1996; Zhang et al., 2010). As a prerequisite of pollination, anther opening has been widely studied with regard to the mechanical and ecophysiological mechanisms of dehiscence (Buchmann, 1983; Keijzer, 1987; Pacini, 2000; Carrizo García et al., 2006). Selective anther opening may respond to pollination environment and enhance pollen dispersal and survival (Castellanos et al., 2006; Passarelli and Cocucci, 2006).
∗ Corresponding authors. Tel.: +86 27 87510526; fax: +86 27 87510526. E-mail addresses: [email protected] (Q.-F. Wang), [email protected] (C.-F. Yang). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2012.01.003
In general, anther opening is a result of tissue desiccation (Schmid, 1976; Buchmann, 1983; Keijzer, 1987). However, it is noteworthy that the dehiscence of the anther can be caused by either evaporation (Keijzer, 1987; Bonner and Dickinson, 1990; Keijzer et al., 1996; Matsui et al., 1999) or reabsorption (Keijzer et al., 1987; Carrizo García et al., 2006). Anther opening due to evaporation or reabsorption must have different responses to relative humidity (RH) and this may potentially affect adaptation of the pollination system. Anther dehiscence due to evaporation must rely to a greater degree on environmental factors while that due to reabsorption is likely to be more dependent on the plant’s own physiological response to environmental change. For example, at high RH, reabsorption, rather than evaporation may occur and result in anther opening. Adaptive linkages between anther characteristics and pollination system have been reported in some taxa, such as Penstemon (Castellanos et al., 2006) and Solanum (Carrizo García and Barboza, 2006; Passarelli and Cocucci, 2006). Antherwall structure affecting pattern of pollen release was found to be correlated with pollination syndrome.
28
J. Li et al. / Aquatic Botany 99 (2012) 27–33
Fig. 1. Characteristics of inflorescence, flower and anther for B. umbellatus. (A) Sequential blooming of flowers (with long pedicel) within an inflorescence (with long stalk); (B–E) asynchronous dehiscence of anthers within a flower: (B) nine undehisced anthers within a flower; (C) early dehiscence of anthers of the outer whorl; (D) anthers of the inner whorl dehiscence later; (E) dehiscence of all the anthers within a flower; stigmas disjoined and becoming receptive; (F) anthers re-closing after water-spraying treatment. Scale bars: 1 cm.
In this study we found that for the aquatic plant Butomus umbellatus L. (the flowering rush) anther opening via longitudinal dehiscence can be reversed when the anther is wetted as happens during rainy weather. Additionally, the flower is dichogamous and the duration of the male phase is quite short. To ascertain the structural and ecophysiological mechanisms of this novel bidirectional process and its possible adaptive significance, experiments were conducted to investigate the following: (1) structural changes of the anther wall during opening and closing; (2) the pattern of environmental factors affecting anther opening and closing both for an individual anther and for the anthers within a flower; (3) change in pollen viability during anthesis and the effect of anther re-closing on pollen viability; and (4) the link between pollinator and anther behaviour.
Pollination observations were conducted in natural populations over 120 h in July 2006, July to August 2007 and July 2009 in which we recorded pollinator type and frequency for each individual plant during rainy episodes, during the intervals (longer than 30 min) between the rainy episodes, as well as during sunny periods. Pollination frequency was estimated as the cumulative period of time spent by pollinators on an umbel in a 1-h observation period. Ten randomly-selected umbels were used during the peak blooming period to calculate the average pollination frequency. Pollinators were also captured during their visits to the flowers and taken to the laboratory for identification. To check for potential autogamy or apomixis, 20 bagged flowers were marked and their fruits later harvested to assess seed set. 2.2. Factors influencing anther behaviour
2. Materials and methods 2.1. Study species B. umbellatus is an emergent, aquatic monocot in a monotypic family that inhabits shallow water around the margins of lakes and slow-moving rivers. Inflorescences are cymose umbels borne on thin, cylindrical, upright stalks that consist of 15–50 pink flowers (Fig. 1A). Flowers contain nectaries and each consists of six conduplicate carpels. The androecium of each flower consists of nine stamens (in an outer whorl of six and an inner whorl of three) (Sun, 1992; Bhardwaj and Eckert, 2001; Fig. 1). Our study was conducted on a natural population at the Wild-Lotus-Garden (45◦ 30 N, 131◦ 51 E), Mishan county, Heilongjiang Province, China during July 2006, July to August 2007, and July 2009. Fifty plants were randomly selected and enclosed with fine bridal veil netting to exclude pollinators. The plants were used to estimate the seed production in the absence of pollinators as well as to provide pollen grains for experiments of pollen stainability since pollen removal by pollinators on fine weather days was quite rapid in the study populations.
Opening flowers were randomly selected at 0800 h before anthers dehisced for observation of anther behaviour. We first recorded the sequence and duration of anther opening within a flower. To observe the detailed behaviour of anthers under conditions of wetness, individual flowers with dehisced anthers were sprayed with rainwater. An atomizer was used to spray each individual flower during a sunny day to simulate rain. The anthers were then photographed to record changes in their structure during water loss on the flower. To investigate the relationship between corolla status (wet/dry) and anther behaviour, we recorded the proportion of closed/re-opened anthers out of the total number of anthers which had dehisced for flowers with dry or wet corollas respectively during sunny periods. Additionally, to evaluate the correlation between corolla status and pollinator visitation, we recorded the percentage of visited flowers out of the total number of flowers with dry and wet corollas respectively during sunny periods. In order to better understand the environmental cues that control the opening and re-closing of anthers, at least 30 freshly opened flower buds (randomly selected on ten individual plants) were
J. Li et al. / Aquatic Botany 99 (2012) 27–33
subjected to variable relative air humidity (RH), low temperature and low light intensity (darkness), respectively. The condition of low temperature was simulated using a transparent plastic bucket containing ice while low light intensity was simulated by fitting a bucket with a cover. Four levels of RH (20%, 50–55%, 70–75%, and 95%) were used in this study. Two levels of RH were created following the methods of Winston and Batesm (1960) using saturated salt solutions in a box covered with transparent plastic film (20% RH with potassium acetate and 95% RH with potassium dichromate). The other two RHs (50–55%, 70–75%) represent the normal ranges of variation in RH in the laboratory and outdoors at the lakeside, respectively at the study site from 0830 to 1700 h on a sunny day. Anther behaviour was observed under the four levels of RH from opened flower buds which were picked at 0800 h and placed in water in vials covered with plastic film. The pedicles were inserted through small holes in the plastic film. Vials with flower buds were placed under the different conditions for observation of anther behaviour. Further in anticipation of potential differences in anther behaviour under conditions of high RH (as occur on rainy days) between anthers in situ and those in vitro, two plants were temporarily enclosed with transparent plastic film to create the condition of high RH (90–95%). Flowers on the two plants were marked at 0800 h before the onset of anther dehiscence and used for observing anther behaviour. 2.3. Structural aspects of anther behaviour To investigate the structural changes of the anther wall during anther opening and re-closing, flower buds and anthers in various states of opening (closed, opened, intermediate, and reclosed) from natural populations were collected and immediately fixed in FAA solution (formalin:acetic acid:70% ethanol at a ratio of 5:6:89 by volume) for laboratory analyses. The surfaces of the anther epidermis (outer wall) were observed by scanning electron microscopy (SEM). Fixed anthers for SEM were subjected to a dehydration series to 100% ethanol, and then dried in CO2 to the critical point, coated with gold, and observed using a Hitachi S-450 SEM at 20 kV. Anthers in different stages were also used to make sections for observation under light microscope. The fixed anthers were dehydrated in a graded ethanol series and then left overnight in 100% ethanol. The material was then passed through graded ethanol/xylene mixtures (100% ethanol; 1:1; and 100% xylene) and embedded through graded xylene/paraffin mixtures (1:1; 100% paraffin). Serial sections (2 m) were obtained with a microtome and stretched on glass slides, then exposed to xylene–ethanol series to remove paraffin and stained with 0.1% safranin.
29
percentage of stained pollen grains out of the total number of pollen grains on a slide. The average for the three samples was regarded as the pollen stainability of an individual flower. To measure the duration of pollen viability, pollen stainability was checked at 0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24, and 36 h after anther dehiscence. To compare pollen viability from exposed and re-closed anthers, dehisced anthers from ten flowers (each from separate bagged plants) were maintained in a re-closed status until 7, 8, 12 and 24 h after anther opening. To maintain anthers in the re-closed status, flowers were treated to intermittent water spraying using an atomizer. Pollen grains for estimation of pollen stainability were obtained from the anthers when they re-opened. To assess the effect of wetting on pollen stainability (and hence viability), fresh pollen grains from 20 flowers selected at random from ten individuals were dipped in water for 30 min and then stained. Since a correlation between fluorochromatic reaction or germinability in vitro and overall pollen competence may be absent (Thomson et al., 1994), we estimated the ability of pollen to germinate on fresh stigmas for pollen grains under different treatments in comparison with control group. Pollen grains from exposed and enclosed anthers on the same plant were applied to different fresh stigmas within a flower 12 and 24 h after anther opening, respectively. To do this, individual stigmas from enclosed flowers at the beginning of the female phase was numbered sequentially and hand-pollinated with the different pollen grains using a brush. Hand pollination was conducted using fresh pollen grains as well as using pollen grains from the same flower but which had been dipped in water. Ten hours after pollination, stigmas with different pollen load were collected and fixed immediately in FAA solution. To estimate pollen performance on stigma, stigmas were observed under a fluorescence microscope (Nikon 80i) after treatment with 8 mol L−1 NaOH for 12 h, followed by dyeing in 0.1% aniline blue. For each treatment, at least 30 pistils were used to estimate pollen performance on the stigmas. A stigma with successful pollen germination was defined as one that recorded 10 or more germinated pollen grains. For pollen and ovule counts, 20 open flowers with no dehisced anthers from 20 randomly selected plants were fixed in FAA solution for laboratory examination following the methods of Yang and Guo (2004). The pollen–ovule ratio was calculated by dividing the total number of pollen grains (nine anthers) by the total number of ovules produced in a flower (six pistils). Data in the study are shown as mean ± S.D. One-way ANOVA was used to compare the changes of pollen stainability under different treatments.
2.4. Pollen viability and pollen–ovule ratio
3. Results
Pollen stainability is commonly used as an indicator of pollen viability (Dafni and Firmage, 2000). In this study we considered stainability of pollen with 2,5-diphenyl tetrazolium bromide (MTT) as an indication of pollen viability. MTT detects the presence of dehydrogenase in viable pollen (Rodriguez-Riano and Dafni, 2000; Zhang et al., 2010). To assess the ability of the technique to discriminate between viable and non-viable pollen, we also used the dye on heat-treated (boiled in water at 100 ◦ C for 2 h) pollen. In the present study, pollen stainability was used for estimating three aspects of pollen viability viz. duration of viability, and differences in viability of pollen from exposed and re-closed anthers and when dipped into water or not. For each treatment, pollen grains from ten flowers on bagged individuals were used. Pollen grains were collected from all anthers within a flower and randomly deposited on three slides (at least 1000 pollen grains per slide) using a fine brush for pollen staining. Estimation of pollen stainability followed the methods of Zhang et al. (2010). Pollen stainability was calculated as the
3.1. Floral biology Sexual reproduction in the flowering rush is dependent on pollen transfer by pollinators. Flowers which were excluded from pollinators set no seed. Eristalis species were the most frequent pollinators. In general, the release of pollen grains relied solely on mechanical contact with the pollinator rather than on shaking of the anther or on gravity. Results showed that pollinator frequency was high (21.6 ± 3.5, N = 36) on sunny days, moderately high (4.2 ± 2.1, N = 28) during the intervals between the rainy episodes (intervals longer than 30 min), and close to zero during rainy episodes. Results of pollinator visitation following the waterspraying treatment showed that corolla wetting clearly affected pollinator behaviour. Flowers with dry corollas received more visitation (90%, N = 96) than those with wet corollas (8%, N = 78). In fine weather, an anther dispersed its entire pollen grains within 3 h of opening while the entire pollen grains in a flower
30
J. Li et al. / Aquatic Botany 99 (2012) 27–33
were removed within 12 h. Under natural conditions, the six aggregate pistils separate at about 12 h after the end of the male function. Stigmas then expand and become receptive sequentially within 6 h. The ivory-colored stigmas become dry and lackluster after 5–7 h by which time they have received pollen grains (Fig. 1A and E). Under natural conditions flower longevity of the species in our study area is about 72 h. 3.2. Factors influencing anther behaviour Within a flower, the anthers presented pollen grains sequentially from 0800 to 1830 h (Fig. 1B–E). No stomata occur on the anther and fresh anthers do not open at night. During anthesis, the six anthers in the outer whorl exposed pollen grains earlier than the three anthers in the inner whorl (Fig. 1C and D). Generally, the average interval between opening of the last anther of the outer whorl and the first anther of the inner whorl is slightly longer than that between the opening of the first and the last anthers within a whorl (see also Fig. 1D). Breakage of the stomium of endothecium in all the anthers within a flower was complete by the time the flower opened. Sequential opening of individual anthers was due to variation among anthers in time for rupture of epidermal cells. Endotheciums of all 63 anthers from opening flower buds were broken. Epidermis cells at the front of stomium in 47 (74.6%) of the anthers were intact while those in the other 16 (25.4%) anthers were ruptured in readiness for the outward bending of the anther wall. No difference was detected in the ability of the anther to re-close between the anthers of the inner and those of the outer whorls in a flower (Fig. 1F). At 20% RH, anther opening (thus exposing pollen grains) was slightly accelerated for the inner whorls but not for the outer whorls. At this level of RH all anthers within a flower had presented their pollen grains before 1500 h. The same pattern was found at 50–55% RH. However, at 95% RH, the time for exposure of pollen grains in both whorls was delayed. At this level of RH approximately 16.0% of the anthers in the outer whorl and approx. 42.2% of the anthers in the inner whorl had no exposed pollen grains by 2000 h. However, for anthers in situ at 90–95% RH, nearly all anthers had presented pollen grains before 1900 h. Furthermore, for unopened in vitro anthers at this RH level the epidermis cells of the stomium had split before 1700 h. When the covering was removed causing RH to drop to 70–75% (environmental RH), the anthers opened within 30 min. Experiments indicated that a one-time treatment of low temperature, high humidity or low light intensity (darkness) did not cause dehisced anthers to re-close. When low temperature and high humidity treatments were combined the resultant condensation created water drops which caused the wetted anthers to re-close. In the simulated rain (spraying) treatment opened anthers re-closed within 4–7 min (5.4 ± 0.8; Fig. 1F). The proportion of re-opened anthers was significantly higher for dry corollas (85%, N = 584) than for wet corollas (11%, N = 389). Closed anthers re-opened within 13–20 min (16.3 ± 2.5) after the corollas dried. The time taken for closed anthers to re-open increased with increase in RH until at 95% RH (for anthers in vitro) it became very infrequent for closed anthers to re-open. 3.3. Structural aspects of anther behaviour Anther opening in B. umbellatus occurs by a longitudinal slit in the side of the anther. The anther wall consists of an epidermis and an endothecium. The later structure has cells with U-shaped wall thickenings (Fig. 2A–C). However, the cells of endothecium in the septa in both locules lack these wall thickenings (Fig. 2A). Unlike the rest of the epidermal cells which enlarged considerably, the epidermal cells facing the septum remained small (Fig. 2A). The breakage
of endothecium in the stomium started during shrinking of the septum. This was followed by opening of the anther after detachment of the adjacent epidermis cells on both sides of the stomium. Once the anther was opened along its entire length, the walls started to shrink and bend outwards. The ventral walls bend until they touch each other, while the dorsal walls bend only partially forming a concave surface. Anther length was noticeably shortened by the outward bending (Fig. 1C and D). The process of anther opening starting from rupture of the epidermis cells to the complete bending outwards of the anther wall could be reversed when the anthers were wetted by spraying with water or by rainfall. Photographs under SEM indicated that the retractile cells of epidermis alternated from the orbicular to the crinkly status when anthers re-closed and re-opened respectively (Fig. 2D and E). Results indicated that epidermis cells were flattened in tangential view and the cells of endothecium were stacked in transverse view when anthers were closed (Fig. 2B). As the anthers opened (re-opened), epidermis cell were compact and their tangential length was noticeably shortened while the cell of endothecium were crinkled and their transverse length was noticeably reduced (Fig. 2C). 3.4. Pollen viability and pollen–ovule ratio Pollen of the species is monosulcate and coated with very little pollenkitt. Results of MTT staining indicated that pollen grains maintained the highest pollen viability during the first 7 h, and then gradually lost viability until they were no longer viable 24 h after anther dehiscence (Fig. 3). Viability decreased rapidly within 30 min in pollen which had been dipped in water (from 57% ± 7% to 14% ± 6%, respectively). However, in the water-spraying experiment pollen grains in re-closed anthers maintained high viability for 12 h which decreased to moderately high viability by 24 h after anther dehiscence (Fig. 3). Moreover, for pollen grains which had dipped in water for 30-min or exposed for 12 h or longer on a dehisced anther, it was difficult to detect any pollen germination on stigma. For stigmas with exposed pollen grains, one out of 31 stigmas (3%) was found to have successful pollen germination after 12 h of exposure. No germination was detected after 24 h of exposure. Results also indicated that successful pollen germination on stigma following pollination with pollen which had been dipped in water for 30-min was low (21%). In contrast, successful pollen germination was found on all stigmas with fresh pollen load. However, the proportion of stigmas with successful pollen germination for enclosed pollen grains from re-closed anthers after 12 and 24 h was relatively high (87% at 12 h and 53% at 24 h respectively). The nine anthers and six pistils of a single flower of B. umbellatus produce on average 4.26 × 105 pollen grains (N = 20, S.D. = 0.54 × 105 ), and 918.1 ovules (N = 20, S.D. = 247.2) respectively. The pollen–ovule ratio in the species was 463.9. 4. Discussion To accomplish pollination, aquatic plants have evolved special mechanisms adapted to water environment (Cox, 1988). For some species of Hydrocharitaceae with hydrophilous pollination, including Vallisneria spiralis and Enhalus acoroides, anthers or flowers carry dry pollen across the water surface to receptive stigma. For the entomophilous B. umbellatus, the unique mechanism of reversible anther opening is of adaptive meaningfulness. Anther behaviour in the species is adapted to the pollination environment and thus enhances the male fitness. 4.1. Anther behaviour Although the mechanism of anther dehiscence and the physiological processes involved in the process are different in different
J. Li et al. / Aquatic Botany 99 (2012) 27–33
31
Fig. 2. Structural aspects of anther in different status. (A–C) Cross sections (sides of epidermis and endothecium are indicated by E and H, respectively): (A) mature undehisced anther without dehiscence; note U-shaped thickenings in cells of endothecium (blue arrow), no U-shaped thickenings in cells of septum (short black arrow), and small epidermis cells at the front of septum (to forming stomium, long black arrow); (B) closing (re-closing) anther wall; note flattened epidermis cells in tangential view and columniform cells of endothecium in transverse view (blue arrow indicating U-shaped thickenings in cells of endothecium); (C) opening (re-opening) anther wall; note compact epidermis cells and squeezed cells of endothecium; (D and E) SEM photograph of epidermis: (D) closing (re-closing) anther; note orbicular epidermis cells; (E) opening (re-opening) anther; note crinkly epidermis cells. Scale bars: A, 100 m; B and C, 20 m; and D and E, 10 m.
species, studies have shown that the structural basis of anther dehiscence in longitudinally dehiscing anthers follows the process summarized by Keijzer (1987) and reported by Carrizo García et al. (2006) as well as in our present study. The finding that anthers in freshly opening flower buds already had endothecium of the their stomium broken but that they were separated in time for epidermis detachment and the outward bending of anther wall is an important finding demonstrating the
asynchronous pattern in this natural process. The results indicated that the three sequential steps of anther opening may be regulated differently in relation water control (see also Carrizo García et al., 2006 for Allium triquetrum). First, stomium breakage is likely a developmentally programmed process related to the development of wall thickenings (Mitsuda et al., 2005). Following the formation of the wall thickenings stomium breakage appeared to be independent of environmental RH. Second, epidermis detachment must be
Fig. 3. Pollen viability of B. umbellatus indicated by MTT staining between exposed pollen grains and pollen grains in closed anthers. Sites with different letter are significantly different at level P < 0.001.
32
J. Li et al. / Aquatic Botany 99 (2012) 27–33
under the control of the plant’s internal physiological mechanisms as evidenced by the observation that the epidermis cells of all the anthers detached once a particular time was reached irrespective of the RH values. Third, unlike the other two stages of anther opening, the outward bending of the anther wall is mainly controlled by moisture levels since higher or lower RH values inhibit or facilitate the process. As may be expected, the changes in anther behaviour in response to environmental moisture levels were more likely to occur at this stage. The mechanism and process of reversible anther opening has been rarely studied since anther opening was always regarded as a unidirectional process (but see Edwards and Jordan, 1992; Wang et al., 2009). In B. umbellatus, this bidirectional process might be ascribed mainly to the retractile epidermis cells. We hypothesize that when the anther is dehydrated, the cells of the epidermis become crinkly and squeeze the endothecium cells, which results in shortening of the epidermis cells and a reduction in the transverse length of the endothecium cells. Consequently, an outward centrifugal force is generated which causes bending of the anther wall. Conversely when the anther rehydrated, a centripetal force arising from the returning of the epidermis and endothecium cells to their pre-dehydration state causes the anther wall to bend inwards (see also Vasil, 1967; Gerenday and French, 1988). Anthers in B. umbellatus opened (re-opened) at different levels of RH. This may indicate that evaporation and reabsorption operate in concert. Evaporation leads to low levels of RH accelerating anther opening (re-opening); however, the opening (re-opening) of the anthers which occurs at high RH suggests that reabsorption must play a role in the processes. Reabsorption may happen more easily for species with long filaments, pedicels, or inflorescence stalks in which water can be transferred (see also Keijzer et al., 1987; Carrizo García et al., 2006). For B. umbellatus, the conspicuously long pedicels and inflorescence stalks (Fig. 1A) may enhance reabsorption. Reabsorption rather than evaporation regulating anther opening is of greater adaptive significance for aquatic plants since RH in the natural habitats of these plants is commonly high. Evaporation has always been regarded as the main cause of the outward bending of anther wall in anther opening (Keijzer et al., 1996; Carrizo García et al., 2006). In the present study, the observation that re-closed anthers in vitro did not re-open even at high RH levels but that in situ the anthers re-opened at 95% RH may indicate that reabsorption rather than evaporation regulated the outward bending of anther wall. In view of the key role of reabsorption in reversible anther opening for aquatic plants, we hypothesize that epidermis cells are hydrophilic and at high RH they absorbed water readily which resulted in re-closing of the anther. Since the anthers have no stoma the water in them is transferred to tissues of adjacent structures (pedicel and stalk) when these tissues were not saturated. This caused the anthers to re-open. This may provide an explanation as to why anthers did not open (re-open) during the night and in rainy weather, but they did so under conditions of high RH. During night time it was difficult to transfer the water in the anther to adjacent tissues owing to the absence of photosynthesis (hence low concentration of solutes) in the plant. Similarly the saturated status of the tissues of the pedicel and stalk during rainy weather hampered transfer of water from the anthers and thus prevented the anthers from opening (re-opening). Under the water-spraying treatment, anthers of flowers with wet corollas did not open (re-open) possibly due to the saturated status of adjacent tissues which hampered transfer of water from the anthers. 4.2. Reversible anther opening enhances male fitness Reversible anther opening has been reported in very few species and is limited to taxa with extended flower longevity such as Lilium philadelphicum (longer than 9 d) (Edwards and Jordan, 1992) and
Paris polyphylla var. yunnanensis (longer than 20 d) (Wang et al., 2009). From the viewpoint of adaptation, species with short-lived flowers or high pollination efficiency do not require to evolve the mechanism of reversible anther opening (see also Edwards and Jordan, 1992). For the dichogamous species B. umbellatus, the duration of male phase was quite short and the efficiency of pollen removal was relatively high owing to the exposed pollen presentation and high pollination frequency (see also Bhardwaj and Eckert, 2001). The unexpected finding of reversible anther opening in this species enriched our understanding of evolution of anther biology. Clearly, the significance of reversible anther opening was enhancement of male fitness by prolonging male duration or avoiding unfavorable environment. Reversible anther opening must achieve this in two ways: Firstly, re-closing anthers help to retain pollen viability since pollen grains in these anthers must take a longer time before they are deposited on stigmas. Secondly, pollinator behaviour must be matched with anther behaviour to ensure relatively rapid pollen export. B. umbellatus pollen may be categorized as partially dehydrated, orthodox type pollen according to its desiccation tolerance (longer than 6 h) and monosulcate shape (Nepi et al., 2001, 2010; Zhang et al., 2010; Franchi et al., 2002, 2011). In the present study, results of pollen staining with MTT indicated that anther re-closing caused pollen to remain viable for a relatively longer period. The maintenance of pollen viability in re-closed anthers might be due to the fact that the re-closed anther created a microenvironment with favorable RH and temperature (see also Stanley and Linskens, 1974; Faegri and van der Pijl, 1979). In addition, the re-closing of the anther avoided waste of pollen grains because pollen grains on an exposed anther may be easily washed away under heavy rain. The phenomenon ensured rapid change of anther behaviour in response to fluctuating weather conditions ensuring the quality of pollen export and prolonging the duration of male phase. In the study area the flowers of B. umbellatus attracted eight insects. The exposed pollen grains are clump in shape due to stickiness and easily adhered to insects visiting the flowers (see also Fernando and Cass, 1997). The efficiency of pollen export for the species was quite high (see also Bhardwaj and Eckert, 2001). However, even at high level of RH during interval of showers, high records of instances of pollinator visitation were made because of the broad pollinator fauna in the species (see also Fernando and Cass, 1997). Correspondingly, re-closed anthers would re-open when the interval between two rainy episodes was longer than 30 min. Results rain simulation (spraying) treatment on sunny days indicated that flowers with wet corolla did not re-open their anthers and they also attracted no pollinators. As evaporation (reabsorption) of water on the corolla occurred, pollinators visited the flowers and the re-closed anthers on the flowers re-opened. According to the criterion of Cruden (1977), the breeding system of B. umbellatus falls into the category between facultative autogamy (168.5) and facultative xenogamy (796.6) with a pollen–ovule ratio of 463.9. Clearly, the dichogamous species B. umbellatus belongs to category of xenogamous taxa. In comparison to other xenogamous species, the low pollen–ovule ratio indicates higher male fitness in the species (Queller, 1984). However, Fernando and Cass (1997) suggested that the developmental irregularities such as meiotic aberration, pollen abortion, excessive winding of pollen tubes and rapid decrease of pollen viability might reduce male reproductive potential. There should be compensatory mechanism enhancing male potential because the sequential anthesis within an inflorescence and staggered anther dehiscence within a flower may increase the chances of pollen to be transferred (Fernando and Cass, 1997). Furthermore, plants of B. umbellatus flowered during the rainy season in Northeastern China (summer rainfall accounted for 90% of the annual total). The remarkable mechanism of reversible anther opening confers on the plant adaptation to the frequent
J. Li et al. / Aquatic Botany 99 (2012) 27–33
scattered showers common at this time of year. Discontinuous pollen presentation effectively prolonged the duration of male phase and avoided unfavorable pollination environment. The unique anther behaviour observed in B. umbellatus might, to a certain extent, by way of enhancing male fitness responsible for the disproportional relationship between pollen–ovule ratio and breeding system in the species (see also Armstrong, 1992; Edwards and Jordan, 1992; Han et al., 2008). The mechanism of reversible anther opening in response to rain may be ascribed mainly to the retractile epidermis cells of anther wall. Reabsorption clearly plays important role in anther opening and re-opening in this aquatic plant with long pedicle and inflorescence stalk. This novel mechanism significantly prolonged pollen longevity by maintaining high pollen viability. The evolving of reversible anther opening in this dichogamous plant with a relatively short flower span is a rather unexpected occurrence. Anther itself controlling pollen dispersal and creating discontinuous pollen presentation avoided unfavorable pollination environment and pollen loss and enhanced pollen competence, thus making the plant better adapted to pluvious flowering season and enhancing male fitness. Acknowledgements We thank Jan Vermaat and two anonymous reviewers for their helpful suggestions and comments, Jin-Ming Chen and Zhi-Yuan Du for assisting in fieldwork, Zhi-Xin Zhu for preparation of the paraffin sections, Xiao-Lin Zhang for MTT staining, Qian Yu for valuable discussions, and Hong Liu (Institute of Zoology, CAS) for assisting in insect identification. This work was supported by the National Natural Science Foundation of China (Grants 31070206 to C.-F.Y and 31170214 to Y.-H.G). References Armstrong, J.E., 1992. Lever action anthers and the forcible shedding of pollen in Torenia (Scrophulariaceae). Am. J. Bot. 79, 34–40. Bernhardt, P., 1996. Anther adaptations for animal pollination. In: D’Arcy, W., Keating, R. (Eds.), The Anthers: Form, Function and Phylogeny. Cambridge University Press, New York, pp. 192–220. Bhardwaj, M., Eckert, C.G., 2001. Functional analysis of synchronous dichogamy in flowering rush, Butomus umbellatus (Butomaceae). Am. J. Bot. 88, 2204–2213. Bonner, J.L., Dickinson, H.G., 1990. Anther dehiscence in Lycopersicon esculentum Mill. II. Water relations. New Phytol. 115, 367–375. Buchmann, S.L., 1983. Buzz pollination in angiosperms. In: Jones, C.E., Little, R.J. (Eds.), Handbook of Experimental Pollination. Van Nostrand Reinhold, New York, pp. 73–113. Carrizo García, C., Barboza, G.E., 2006. Anther wall development and structure in wild tomatoes (Solanum sect. Lycopersicon): functional inferences. Aust. J. Bot. 54, 83–89. Carrizo García, C., Nepi, M., Pacini, E., 2006. Structural aspects and ecophysiology of anther opening in Allium triquetrum. Ann. Bot. 97, 521–527. Castellanos, M.C., Wilson, P., Keller, S.J., Wolfe, A.D., Thomson, J.D., 2006. Anther evolution: pollen presentation strategies when pollinators differ. Am. Nat. 167, 288–296. Cox, P.A., 1988. Hygrophilous pollination. Annu. Rev. Ecol. Syst. 19, 261–280. Cruden, R.W., 1977. Pollen–ovule ratios: a conservative indicator of breeding systems in flowering plants. Evolution 31, 32–46.
33
Dafni., A., Firmage, D., 2000. Pollen viability and longevity: practical, ecological and evolutionary implications. Plant Syst. Evol. 222, 113–132. Edwards, J., Jordan, J.R., 1992. Reversible anther opening in Lilium philadelphicum (Liliaceae): a possible means of enhancing male fitness. Am. J. Bot. 79, 144–148. Faegri, K., van der Pijl, 1979. The Principles of Pollination Ecology, 3rd ed. Pergamon Press, Oxford. Fernando, D., Cass, D.D., 1997. Developmental assessment of sexual reproduction in Butomus umbellatus (Butomaceae): male reproductive component. Ann. Bot. 80, 449–456. Franchi, G.G., Nepi, M., Dafni, A., Pacini, E., 2002. Partially hydrated pollen: taxonomic distribution, ecological and evolutionary significance. Plant Syst. Evol. 234, 211–227. Franchi, G.G., Piotto, B., Nepi, M., Baskin, C.C., Baskin, J.M., Pacini, E., 2011. Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival. J. Exp. Bot. 62, 5267–5281. Gerenday, A., French, J.C., 1988. Endothecial thickenings in anthers of porate monocotyledons. Am. J. Bot. 75, 22–25. Han, Y., Dai, C., Yang, C.-F., Wang, Q.-F., Motley, T.J., 2008. Anther appendages of Incarvillea trigger a pollen-dispensing mechanism. Ann. Bot. 102, 473–479. Harder, L.D., Thomson, J.D., 1989. Evolutionary options for maximizing pollen dispersal of animal pollinated plants. Am. Nat. 133, 323–344. Keijzer, C.J., 1987. The processes of anther dehiscence and pollen dispersal. I. The opening mechanism of longitudinally dehiscing anthers. New Phytol. 105, 487–498. Keijzer, C.J., Hoek, I.H.S., Willemse, M.T.M., 1987. The processes of anther dehiscence and pollen dispersal. III. The dehydration of the filament tip and the anther in three monocotyledonous species. New Phytol. 106, 281–287. Keijzer, C.J., Leferink-ten Klooster, H.B., Reinders, M.C., 1996. The mechanics of the grass flower: anther dehiscence and pollen shedding in maize. Ann. Bot. 78, 15–21. Matsui, T., Omasa, K., Horie, T., 1999. Mechanism of anther dehiscence in rice (Oryza sativa L.). Ann. Bot. 84, 501–506. Mitsuda, N., Seki, M., Shinozaki, K., Ohme-Tagaki, M., 2005. The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17, 2993–3006. Nepi, M., Cresti, L., Guarnieri, M., Pacini, E., 2010. Effect of relative humidity on water content, viability and carbohydrate profile of Petunia hybrida and Cucurbita pepo pollen. Plant Syst. Evol. 284, 57–64. Nepi, M., Franchi, G.G., Pacini, E., 2001. Pollen hydration status at dispersal: cytophysiological features and strategies. Protoplasma 216, 171–180. Pacini, E., 2000. From anther and pollen ripening to pollen presentation. Plant Syst. Evol. 222, 19–43. Passarelli, L., Cocucci, A., 2006. Dynamics of pollen release in relation to anther-wall structure among species of Solanum (Solanaceae). Aust. J. Bot. 54, 765–771. Queller, D.C., 1984. Pollen–ovule ratios and hermaphrodite sexual allocation strategies. Evolution 38, 1148–1151. Rodriguez-Riano, T., Dafni, A., 2000. A new procedure to assess pollen viability. Sex. Plant Reprod. 12, 241–244. Schmid, R., 1976. Filament histology and anther dehiscence. Bot. J. Linn. Soc. 73, 303–315. Stanley, R.G., Linskens, H.F., 1974. Pollen: Biology, Biochemistry, Management. Springer-Verlag, Berlin. Sun, X.Z., 1992. Flora of China, vol. 8. Science Press, Beijing. Thomson, J.D., Rigney, L.P., Karoly, K.M., Thomson, B.A., 1994. Pollen viability, vigor, and competitive ability in Erythronium grandiflorun (Liliaceae). Am. J. Bot. 81, 1257–1266. Vasil, I.K., 1967. Physiology and cytology of anther development. Biol. Rev. 42, 327–373. Wang, D.K., Sun, G.F., Wang, L.F., Zhai, S.H., Cen, X.J., 2009. A novel mechanism controls anther opening and closing in Paris polyphylla var. yunnanensis. Chin. Sci. Bull. 54, 244–248. Winston, P.W., Batesm, D.H., 1960. Saturated solutions for the control of humidity in biological research. Ecology 41, 232–237. Yang, C.-F., Guo, Y.-H., 2004. Pollen size-number trade-off and pollen–pistil relationships in Pedicularis (Orobanchaceae). Plant Syst. Evol. 247, 177–185. Zhang, X.L., Gituru, R.W., Yang, C.F., Guo, Y.H., 2010. Exposure to water increased pollen longevity of pondweed (Potamogeton spp.) indicates different mechanisms ensuring pollination success of angiosperms in aquatic habitat. Evol. Ecol. 24, 939–953.