removal in hermaphrodite flowers

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1 Functional Ecology 2003 Anther arrangement influences pollen deposition and Blackwell Publishing Ltd. removal in hermaphrodite flowers G. KUDO Graduate School of Environmental Earth Science, Hokkaido University, Sapporo , Japan Summary 1. Effects of anther arrangement on pollen deposition and removal in hermaphrodite flowers were experimentally quantified to assess the ecological significance of stamen structure. The anther arrangement of Brassica rapa was modified into six types with respect to anther number and the spatial arrangement of long and short stamens. Each anther treatment was combined with a nectar-intact and nectar-depleted treatment. After single visits by bumblebees in a glasshouse, the visit duration to each flower and the number of pollen grains deposited on stigmas and pollen grains remaining in long and short anthers were measured. 2. Visits were significantly shorter in nectar-depleted flowers. Short anthers also extended visit duration, but modification of the long anther arrangement had no effect. Pollen deposition on stigmas depended on the number of long anthers and on visit duration. Although an increase in long anther number accelerated self-pollination, removal of short anthers did not influence pollen deposition. 3. Pollen removal from long anthers depended on anther treatment and visit duration, but not on nectar availability. Any modification of anther arrangement tended to increase pollen removal during single visits. Long anthers contributed to the decrease in pollen removal per visit, while pollen removal from short anthers depended only on visit duration. Short anthers appeared to have a different function from the restriction of pollen removal during single visits by bumblebees. 4. Thus, long and short anthers made different contributions to pollination efficiency of nectar-feeding bumblebees. The maintenance of different types of stamens in Brassicaceae plants (tetradynamous stamens) may reflect functional advantages for a diverse pollination syndrome if the pollination efficiency of long and short anthers varies depending on the type of pollinator. Key-words: Brassica rapa, bumblebee, nectar, pollination efficiency, visit duration Functional Ecology (2003) Ecological Society Introduction Many floral traits of hermaphrodite plants have probably evolved by sexual selection acting on male function: successful pollen export to compatible stigmas (Willson 1979; Lloyd & Yates 1982; Queller 1983; Bell 1985; Stanton, Snow & Handel 1986). Previous studies have demonstrated that animal-pollinated plants could maximize pollination success by restricting pollen removal by individual pollinators and exploiting all those that are available (Lloyd & Yates 1982; Harder & Thomson 1989). Floral morphology (size and shape), nectar production, staggered anther dehiscence and anther arrangement influence pollen receipt and dispersal during single visits of pollinators (reviewed by Author to whom correspondence should be addressed. E- mail: gaku@ees.hokudai.ac.jp Harder et al. 2001). Among these characters, anther arrangement the number and position of anthers can influence pollen removal by regulating the likelihood and extent of direct contact with pollinators (Harder 1990; Murcia 1990). Anther arrangement can also determine pollen placement on a pollinator s body, which influences the likelihood of pollen loss through grooming (Fægri & van der Pijl 1979; Harder 1990). Anthers of hermaphrodite plants, especially in species lacking other specific pollen packaging and dispensing mechanisms, should be arranged to maximize the pollen dispersal of individual flowers (Barrett, Cole & Harder 1998). If so, any modification of anther arrangement is likely to decrease successful pollen dispersal during single visits of pollinators. However, the effects of anther arrangement on pollen dispersal are poorly studied, with only a few investigations on heterostylous plants (Wolfe & Barrett 1989; Kohn & Barrett 1992; Harder & Barrett 1993). 349

2 350 G. Kudo Fig. 1. Six arrangements of Brassica rapa anthers: each was composed of a treatment of short (S) anthers (S0 or S1) and a treatment of long (L) anthers (L0, L1 or L2). S0, no short anthers; S1, intact (one pair of short anthers); L0, no long anthers; L1, lacking one pair of long anthers; L2, intact (two pairs of long anthers). In addition to restricted pollen removal per visit, avoidance of pollen stigma interference may be an important effect of anther arrangement (Webb & Lloyd 1986). In self-incompatible species, increased self-pollen deposition may reduce seed set through stigma clogging, while in self-compatible species increased self-pollination could increase selfing rates. Such interference would decrease male and female fitness through pollen discounting and low seed set or production of inferior seeds by inbreeding depression. Therefore not only anther arrangement itself, but also the position of each anther relative to stigma height, would affect the success of pollen dispersal in hermaphrodite flowers. Flowers of Brassicaceae species have a fixed number of stamens and a stereotypical structure, with one pair of short stamens arranged in the outer part of the flower and two pairs of long stamens in the inner part, i.e. tetradynamous stamen condition (Heywood 1993; Zomlefer 1994). I hypothesize that the functional significance of this anther arrangement is to restrict pollen removal through two possible mechanisms: (1) controlling the handling time of pollinators to reach the nectary at the base of each short stamen (Davis et al. 1998); (2) controlling the manner of contact between anthers and a pollinator s body during nectar foraging. Because nectar quality and quantity of individual flowers often influence the duration of pollinator visit, pollen deposition, and removal per visit (Galen & Plowright 1985; Thomson 1986; Mitchell 1993), the effectiveness of anther arrangement may also be influenced by the nectar conditions of individual flowers. It is important to distinguish the effects of anther arrangement and nectar conditions on pollen dispersal in order to test the significance of anther arrangement. To perform such tests, the anther arrangement of self-incompatible Brassica rapa flowers was manipulated. Six types of anther manipulations (including unmanipulated flowers) were conducted with respect to anther number and the spatial arrangement of anthers (Fig. 1). For each type of manipulation, a nectar-depletion treatment and a nectar control were included, and a pair of nectar-treated plants was visited by nectar-feeding bumblebees. I then compared the visit duration of bumblebees and the number of pollen grains deposited on stigmas and pollen remaining in anthers after single visits between the anther types and between the nectar conditions, to test the success of pollen dispersal in individual flowers. The primary objectives of this study were to answer the following questions: (1) does the modification of anther arrangement influence the visit duration of pollinators, pollen removal and pollen deposition in individual flowers? (2) If so, are there any interactions between anther arrangement and nectar production in terms of the pollination process? (3) How do anther number and spatial structure (long and short anthers) affect pollen deposition on stigmas and removal from anthers during single visits? (4) Is there any functional difference between long and short anthers? Materials and methods A rapid-cycling, wild-type stock of Brassica rapa was used, which has been developed by artificial selection for early flowering and small size (Williams & Hill 1986). Plants were grown two per 155 ml pot in the laboratory under continuous illumination by cool-white fluorescent bulbs (230 µmol m 2 s 1 PAR). The bumblebees (Bombus occidentali) came from a captive colony for agricultural use. The box containing this colony was connected to a flight cage ( cm), allowing the bumblebees access to B. rapa plants. Before the experiment, the bumblebees were trained to enter the cage, visit B. rapa flowers and return to their nest box. During the experiment the colony was supplied with pollen, but the supply of nectar was limited to encourage foraging. The experiment considered the joint effects of anther arrangement and nectar availability (intact or depleted) on bumblebee behaviour, pollen removal

3 351 Effects of anther position on pollination and pollen receipt. To create different anther arrangements, 0 or 1 pairs of short anthers (S0 or S1) and 0, 1 or 2 pairs of long anthers (L0, L1 or L2) were removed, creating six categories of plants (Fig. 1: S0L0, S0L1, S0L2, S1L0, S1L1, S1L2). On each day of the experiment, six pots were chosen in which both plants had more than three open flowers that bloomed within 24 h. The two plants in each pot served as a single anther arrangement, but received contrasting nectar treatments. For each plant, all open flowers were removed except the upper three flowers with dehisced anthers (at this flowering stage, stigmas typically extend slightly beyond the long anthers). Anthers (but not filaments) were then removed with fine forceps. Just before using a pair of plants in the experiment, nectar was extracted from the three flowers on one randomly selected plant per pot with small filterpaper wicks. The technique of extracting nectar with filter-paper wicks did not remove all available nectar because of high viscosity under low humidity in the laboratory. Therefore the nectar treatment contrasted unaltered flowers (nectar-intact flowers) with those from which most of the nectar had been removed (nectar-depleted flowers), rather than empty flowers. On each of the 20 days of this experiment (9 April to 10 May 2001), one pot was tested for each of the six anther treatments between 10:00 and 12:00 h. The six treatments were tested in random order each day (12 plants from six pots per day). During an experimental trial, the two experimental plants in a pot were exposed to pollination by a bumblebee and the anthers and stigmas collected immediately. In addition to the experimental plants, each bumblebee had access to at least 20 B. rapa plants with intact flowers to provide pollen for deposition on the stigmas of experimental flowers and to maintain the bumblebee s interest in foraging. When a bumblebee visited the experimental plants, its behaviour was videotaped to record the number of flowers visited and their durations (s). Among 240 visits throughout the experiment, 92 1% were for nectar feeding only; 6 7% were for pollen collection only; and 1 2% were for nectar feeding and pollen collection. Because this analysis considers only trials during which worker bees fed only nectar from flowers that received a single visit, data on pollen-collecting bees, multiple visits and male bees visits were eliminated. As a result, observations for 388 flowers on 200 plants were available for analysis. After the bumblebees left the experimental plants, they were removed from the flight cage and the exposed stigmas and anthers were collected. The style of each flower was excised, mounted vertically with its base in 1% agar on a culture plate, and kept for longer than 6 h under moist conditions to make pollen fully adhere to the stigma surface. The number of pollen grains deposited on each stigma was then counted under a dissecting microscope ( 50) after staining with acetocarmine. The long and short anthers (if present) were also collected separately from each flower using fine forceps, and stored in separate vials containing 70% ethanol. Later, the pollen remaining in anthers was counted electronically with a particle counter (Elzone 5380, Micromeritics, USA; Harder 1990). Before counting, all vials were agitated in an ultrasonic water bath for 15 min to dislodge pollen from the anthers. Statistical analyses of visit duration, pollen deposition and pollen removal employed repeated-measures ANOVA and ANCOVA (SAS version 8 1, Proc Mixed; SAS Institute Inc. 1999). All analyses considered pots with paired plants as experimental subjects, so that the numbers of pairs of short and long anthers were between-subject factors, whereas nectar treatment was a within-subject factor. Some analyses of pollen deposition and removal also considered visit duration as a covariate. The full general linear model for these analyses involving the covariate included all possible interactions. Backward elimination (α = 0 05) of nonsignificant interactions involving the covariate was used to select the models reported here. Because of the repeated measures, restricted maximum likelihood was used to characterize the covariance between responses for plants in the same pot (Jennrich & Schluchter 1986). In all cases, compound symmetry was a more appropriate covariance model than one of independent responses (P < 0 05). Denominator degrees of freedom for F tests in these analyses were calculated by Satterthwaite s approximation (Littell et al. 1996). Because of non-normality and heterogeneous variances, all analyses were log-transformed (visit duration, pollen remaining) or square-root-transformed data (pollen receipt). A posteriori contrasts involving all pairwise comparisons involved Tukey s procedure for controlling the experiment-wise type I error rate to α = 0 05, whereas those involving other families of contrasts used the Dunn Sidák procedure (Kirk 1995). Results VISIT DURATION During the experiment, bumblebees spent relatively long periods visiting B. rapa flowers (mean ± SE = 12 7 ± 0 7 s for nectar-intact flowers and 8 6 ± 0 4 s for nectar-depleted flowers across anther types; Table 1). In general, the long visits probably reflected the low humidity in the laboratory where the plants were grown and the experiment was conducted, which resulted in concentrated, viscous nectar. The duration of visits by bumblebees to B. rapa flowers depended on nectar availability and anther arrangement (Table 2). Given that they foraged for nectar, it is not surprising that bumblebees visited flowers for longer when nectar was not depleted than when it was depleted (Table 1). In addition, the presence of short anthers (S1) significantly increased visit duration (14 2 ± 1 1 s for nectar-intact flowers and 10 1 ± 0 7 s for nectar-depleted flowers) compared to

4 352 G. Kudo Table 1. Visit duration of bumblebees, number of pollen grains deposited per stigma, and pollen grains remaining in each long (L) and short (S) anther after single visits in flowers of Brassica rapa with different anther types and nectar treatments Anther type S1L2 S0L2 S1L1 S0L1 S1L0 S0L0 Visit duration (s) Nectar intact 14 5 ± 1 5 (36) 10 2 ± 1 5 (22) 12 4 ± 1 6 (28) 13 5 ± 2 1 (36) 15 3 ± 2 2 (36) 9 0 ± 1 3 (34) Nectar depleted 9 9 ± 1 3 (29) 8 9 ± 1 1 (36) 11 0 ± 1 6 (25) 7 1 ± 0 8 (38) 9 5 ± 0 9 (34) 6 0 ± 0 8 (34) Pollen deposition per stigma Nectar intact 490 ± ± ± ± ± ± 13 Nectar depleted 415 ± ± ± ± ± ± 14 Pollen remaining per L-anther Nectar intact 2760 ± ± ± ± 93 Nectar depleted 2330 ± ± ± ± 245 Pollen remaining per S-anther Nectar intact 2940 ± ± ± 291 Nectar depleted 2870 ± ± ± 335 Mean ±1 SE. Sample sizes are shown in parentheses. See Fig. 1 for anther types. flowers from which these anthers had been removed (S0; 11 0 ± 1 0 and 7 3 ± 0 5 s, respectively). In contrast, visit duration did not vary significantly with the number of pairs of long anthers in a flower. The absence of significant interactions between factors indicates that the presence of nectar and of short anthers affected visit duration independently of each other and of the number of long anthers. POLLEN DEPOSITION Pollen receipt during single visits by bumblebees varied extensively ( grains across anther types and nectar treatments; Table 1). Some of this variation was associated with the effects of nectar depletion, anther removal and visit duration (Table 2). When the statistical analysis did not consider visit duration, pollen deposition increased significantly with the numbers of long and short anthers, and decreased significantly with nectar depletion. However, neither the number of short anthers nor nectar depletion influenced pollen receipt when the analysis also considered the significant positive effect of visit duration. This difference between the analyses indicated that nectar volume and the presence of short anthers affected pollen receipt indirectly through their influences on visit duration. This result contrasts with the influence of the number of long anthers, which remained significant when the analysis accounted for variation in visit duration. The continual increase in pollen deposition with the number of long anthers (Fig. 2a) probably reflected increasing opportunity for intrafloral self-pollination, as long anthers within a flower are often at the same height as the stigma. Table 2. Results of ANOVA for visit duration, and ANOVA and ANCOVA for pollen receipt by stigmas and pollen remaining in each short (S) and long (L) anthers after single visits to Brassica rapa flowers by bumblebees Pollen receipt Pollen remaining Pollen remaining in L-anthers Effect Visit duration (ANOVA) (ANOVA) (ANCOVA) in S-anthers (ANCOVA) (ANOVA) (ANCOVA) Pairs of S-anthers (S) F 1,107 = 6 01* F 1,102 = 6 11* F 1,105 = 3 01 F 1,75 = 0 15 F 1,75 = 0 87 Pairs of L-anthers (L) F 2,105 = 0 91 F 2,100 = 45 03*** F 2,100 = 46 00*** F 2,47 = 2 42 F 1,68 = 12 23*** F 1,68 = 16 73*** S L F 2,113 = 0 84 F 2,104 = 2 46 F 2,105 = 1 85 F 1,75 = 0 19 F 1,75 = 0 17 Nectar depletion (N) F 1,291 = 30 39*** F 1,300 = 5 07* F 1,316 = 0 98 F 1,159 = 0 73 F 1,187 = 4 73* F 1,194 = 0 81 S N F 1,293 = 0 03 F 1,305 = 0 10 F 1,306 = 0 15 F 1,190 = 0 01 F 1,190 = 0 04 L N F 2,291 = 0 29 F 2,300 = 1 11 F 2,301 = 1 18 F 2,152 = 0 93 F 1,187 = 9 53** F 1,187 = 9 82** S L N F 2,293 = 0 74 F 2,304 = 0 68 F 2,306 = 0 58 F 1,190 = 1 13 F 1,190 = 0 45 ln(visit duration) F 1,320 = 28 19*** F 1,139 = 13 11*** F 1,230 = 32 72*** *P 2003 < 0 05, British **P < 0 01, ***P < Ecological Analysis of Society, pollen remaining in S-anthers considered only treatments with S-anthers (S1L0, S1L1 and S1L2), whereas that for L-anthers considered only Functional treatments Ecology, with L-anthers (S0L1, S0L2, S1L1 and S1L2; see Fig. 1). ANOVA results for pollen remaining in S-anthers are not shown due to non-significant 17, difference for every effect.

5 353 Effects of anther position on pollination the overall removal rate increased with increasing visit duration. The experimental manipulations affected pollen removal from short and long anthers differently (Table 2). Neither nectar nor anther manipulation affected removal from short anthers significantly, regardless of whether visit duration was included in the analysis. In contrast, pollen removal per long anther depended on interacting effects of nectar depletion and the number of long anthers in a flower (Table 2; Fig. 2b). Nectar depletion significantly increased the pollen remaining per long anther (i.e. reduced pollen removal) in flowers with only one pair of long anthers (Dunn Sidák multiple comparison, t 191 = 2 86, P < 0 05), but not in intact flowers (t 190 = 1 53, P > 0 4). In addition, removal of one pair of long anthers significantly reduced the pollen remaining (i.e. increased removal) in flowers with unmanipulated nectar (t 117 = 5 06, P < 0 001), but not in those from which nectar had been depleted (t 111 = 1 97, P > 0 1). This general interaction was not affected by whether visit duration was included in the analysis. Fig. 2. Relationships between nectar depletion (control, ; depleted, ), number of pairs of long anthers and mean (± SE) amounts of pollen deposited per stigma (a); and pollen remaining per long anther (b) after single visits on Brassica rapa flowers. L0, no long anthers (S0L0 and S1L0); L1, one pair of long anthers (S0L1 and S1L1); L2, two pairs of long anthers (S0L2 and S1L2). Sample size is shown in parentheses. POLLEN REMOVAL Single visits by bumblebees removed a large fraction of the pollen produced by B. rapa anthers. The plants produced an average of 6190 ± 231 pollen grains per long anther (n = 129) and 5520 ± 364 grains per short anther (n = 94). Based on the pollen remaining in anthers after a visit (Table 1), bumblebees removed 78% of the pollen from long anthers and 72% from short anthers, averaged over all treatments. Despite the similarity in the overall proportion of pollen removed, the influences on pollen removal differed between the two anther lengths (Table 2). Not surprisingly, the amount of pollen remaining on anthers of both types declined significantly with increasing visit duration (Table 2; partial regression coefficient ± SE: short anthers, ± 0 095; long anthers, ± 0 069). Given that pollen remaining and visit duration were both ln-transformed for these analyses, comparison of the partial regression coefficients with 1 reveals whether the overall rates of pollen removal varied with visit duration. For both anther types, the partial regression coefficients were significantly less than 1 (short anthers, t 139 = 6 93, P < 0 001; long anthers, t 230 = 8 81, P < 0 001), indicating that Discussion Anther arrangement in Brassica rapa influenced pollen deposition and removal during single visits of nectarfeeding bumblebees. However, long and short anthers had different effects on pollinator behaviour and pollination efficiency. While the presence of short anthers significantly increased visit duration, no modification of long anthers influenced the duration of single visits. The arrangement of long anthers appears to regulate pollen dynamics by controlling the manner of contact between the anthers and a pollinator s body (Harder 1990; Murcia 1990; Kohn & Barrett 1992; Harder & Barrett 1993) rather than by controlling the visit duration of single pollinators. These results indicate the functional difference between anther positions. The effects of nectar depletion on pollen deposition and removal disappeared when visit duration was considered. This indicates that the existence of nectar indirectly influenced pollination efficiency through a longer visit duration on flowers with nectar (Galen & Plowright 1985; Thomson 1986). In contrast, anther type directly influenced pollen deposition and removal. The extent of pollen deposition depended on the number of long anthers and visit duration. Flowers with one pair of long anthers had significantly fewer pollen grains remaining in anthers after one visit than did flowers with two pairs of long anthers. Estimated pollen removal rates calculated from data of nectarintact flowers and the number of pollen grains of unvisited flowers were 55% in S1L2, 62% in S0L2, 80% in S1L1, and 86% in S0L1 in long anthers; and 47% in S1L2, 60% in S1L1, and 69% in S1L0 in short anthers. Any modification of anther arrangement increased pollen removal per visit, although the amount of pollen remaining in short anthers did not differ significantly

6 354 G. Kudo among anther types. If restriction of pollen removal during single visits results in a successful pollen export in this species (Lloyd & Yates 1982; Harder & Thomson 1989), anthers of B. rapa may be arranged to maximize pollination success, as hypothesized. Long anthers contributed to restricting pollen removal per visit, but accelerated the self-pollen deposition on stigmas which may cause pollen stigma interference (sensu Webb & Lloyd 1986). How can plants reconcile the structural trade-off between male and female success in the pollination process? Bell & Cresswell (1998) reported that in Brassica napus, residual female function (proportion of ovules available for fertilization) decreased rapidly after opening, while residual male function (proportion of pollen grains remaining in anthers) decreased slowly due to the gradual dehiscence of anthers. In this experiment, flowers with fully dehisced anthers were used. However, such slight differences in phenological schedules between sexual functions may reduce sexual interference (Robertson & Lloyd 1993). In contrast to the effect of the number of long anthers, the spatial arrangement of anthers had no significant effect on pollen deposition or removal. Differences in pollen removal between long and short anthers have been reported in previous studies on tristylous plants (Wolfe & Barrett 1989; Harder & Barrett 1993). Long anthers have generally high pollen removal rates, while removal from short anthers is more stochastic because each type of anther contacts different portions of a pollinator s body. Such a tendency also appears to apply to B. rapa, a monomorphic, hermaphrodite species. Spatial arrangement of anthers may determine the placement of pollen on a pollinator s body, which influences the likelihood of pollen loss through grooming and subsequent pollen carryover (Fægri & van der Pijl 1979; Waser & Price 1984; Wolfe & Barrett 1989). As another significant effect of spatial anther arrangement, the effect of pollinator type may be important. Here, I focused on pollination by nectarfeeding workers of a single Bombus species. However, pollination efficiency should depend on the type of pollinator (specialist vs generalist), its behaviour (nectar feeder vs pollen collector) and body size (Motten et al. 1981; Herrera 1987; Harder & Barrett 1993; Conner, Davis & Rush 1995). In the tristylous Pontederia cordata, for example, pollen from long anthers was largely removed by bumblebees, but a smaller anthophorid bee Melissodes apicata did not touch those anthers during foraging (Harder & Barrett 1993). Their study indicated that the relative importance of anther position depends on the type of pollinator. Differences in various floral characters have been reported between flowers visited by passive pollen collectors (nectar feeders) and by active pollen collectors (reviewed by Bernhardt 1996). Various pollinators, including passive and active pollen collectors, visit flowers of Brassicaceae plants, and pollen removal from long and short anthers varies depending on the type of pollinators, such as bees, butterflies or syrphid flies (Conner, Davis & Rush 1995). Therefore the presence of short stamens may have specific functions for some pollen collectors, other than a barrier against easy access to the nectary for nectar feeders, resulting in a longer stay. Tetradynamous stamens characteristic of Brassicaceae flowers may reflect the functional advantages in pollination efficiency for diverse pollination syndrome. Acknowledgements Special thanks go to Lawrence Harder for his guidance in planning, help with statistical analyses, and critical discussion on this study. 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