Touch-sensitive stamens enhance pollen dispersal by scaring away visitors

Deng-Fei Li; Wen-Long Han; Susanne S Renner; Shuang-Quan Huang

doi:10.7554/eLife.81449

. 2022 Oct 11;11:e81449. doi: 10.7554/eLife.81449

Touch-sensitive stamens enhance pollen dispersal by scaring away visitors

Deng-Fei Li ¹, Wen-Long Han ¹, Susanne S Renner ², Shuang-Quan Huang ^1,^✉

Editors: Bernhard Schmid³, Jürgen Kleine-Vehn⁴

PMCID: PMC9555859 PMID: 36217820

Abstract

Animal-pollinated plants have to get pollen to a conspecific stigma while protecting it from getting eaten. Touch-sensitive stamens, which are found in hundreds of flowering plants, are thought to function in enhancing pollen export and reducing its loss, but experimental tests are scarce. Stamens of Berberis and Mahonia are inserted between paired nectar glands and when touched by an insect’s tongue rapidly snap forward so that their valvate anthers press pollen on the insect’s tongue or face. We immobilized the stamens in otherwise unmodified flowers and studied pollen transfer in the field and under enclosed conditions. On flowers with immobilized stamens, the most common bee visitor stayed up to 3.6× longer, yet removed 1.3× fewer pollen grains and deposited 2.1× fewer grains on stigmas per visit. Self-pollen from a single stamen hitting the stigma amounted to 6% of the grains received from single bee visits. Bees discarded pollen passively placed on their bodies, likely because of its berberine content; nectar has no berberine. Syrphid flies fed on both nectar and pollen, taking more when stamens were immobilized. Pollen-tracking experiments in two Berberis species showed that mobile-stamen-flowers donate pollen to many more recipients. These results demonstrate another mechanism by which plants simultaneously meter out their pollen and reduce pollen theft.

Research organism: Other

Introduction

Animals visit flowers to forage for food or other rewards, mainly nectar or pollen (Ollerton, 2021). From the perspective of male reproductive success, nectar and pollen are entirely different rewards because paternity is maximized if pollen grains from one flower are deposited on multiple conspecific recipients, rather than ending up as food, while nectar is produced as pollinator food (Westerkamp, 1996; Westerkamp and Classen-Bockhoff, 2007). A plant’s success as a father can depend on its temporal deployment of pollen and on the accuracy of pollen placement on the most effective pollen vectors (Harder and Thomson, 1989; Armbruster et al., 2014, and studies cited therein). Flowers are therefore under selection to ‘pay’ visitors as much as possible by nectar, which can usually be replenished, and to meter out their non-replenishable pollen grains by placing them on multiple high-fidelity vectors. Two ways in which plants achieve this are by filtering their visitors, such that pollen consumers are repelled, or by encouraging pollinators to move on, resulting in less pollen and/or nectar consumption per flower while retaining pollen import and export (male and female fitness).

Biologists from Linnaeus (1755) onward have been aware of the forward-snapping movement of the stamens of Berberis vulgaris once the filament base of an individual stamen is touched by a nectar-drinking insect (or a pointed object). Berberis flowers have six petals and six stamens, each inserted between two nectaries. Nectar constitutes the main floral reward. The relatively few pollen grains produced remain hidden in the paired pollen sacs that open by apical valves (Figure 1, Supplementary file 1; pollen/ovule ratios in Berberis are around 2380–3400; this study). Different species of Berberis and Mahonia, a close relative, vary in the rapidity of their stamen movements and also in the extent of recovery and repeatability, but the first movement of single stamen is generally completed in fractions of seconds (Percival, 1965; Lechowski and Białczyk, 1992; Lebuhn and Anderson, 1994; this study).

Figure 1. — The major pollinators, workers of *Apis cerana* (A), and a long-tongued bee, *Habropoda sichuanensis* (B), sucking nectar while their tongues (arrow) may contact filaments, anthers, and/or stigmas. These bees do not groom *Berberis* pollen into their corbiculae, and their legs are therefore without pollen loads (hollow arrows). (C) *Rhingia campestris* feeding on nectar and pollen. A bee visiting two flowers with experimentally immobilized and hence touch-insensitive stamens (D). (E) A cross section of a floral bud, showing the two anther valves and two nectaries at the base of each petal. (F) Natural flower with mobile stamens (left) bending inward when their filament bases are touched by a needle; stamen-immobilized (SI) flower (right) whose pedicel had been immersed in 75% alcohol for over 30 min. (G) Diagram of stamen-mobile and SI flowers, illustrating the stamen movement when a bee’s tongue touches the filament. (H) A floral array on an inflorescence in the field with four alcohol-treated SI flowers (arrows). (I) Stained pollen grains (red) deposited on a stigma under open pollination in the field.

Figure 1—figure supplement 1. — The major pollinators, workers of *Apis cerana* (A), and a long-tongued bee, *Habropoda sichuanensis* (B), sucking nectar while their tongues (arrow) may contact filaments, anthers, and/or stigmas. These bees do not groom *Berberis* pollen into their corbiculae, and their legs are therefore without pollen loads (hollow arrows). (C) *Rhingia campestris* feeding on nectar and pollen. A bee visiting two flowers with experimentally immobilized and hence touch-insensitive stamens (D). (E) A cross section of a floral bud, showing the two anther valves and two nectaries at the base of each petal. (F) Natural flower with mobile stamens (left) bending inward when their filament bases are touched by a needle; stamen-immobilized (SI) flower (right) whose pedicel had been immersed in 75% alcohol for over 30 min. (G) Diagram of stamen-mobile and SI flowers, illustrating the stamen movement when a bee’s tongue touches the filament. (H) A floral array on an inflorescence in the field with four alcohol-treated SI flowers (arrows). (I) Stained pollen grains (red) deposited on a stigma under open pollination in the field.

Early workers thought that the unidirectional stamen movements to the flower center, where the stigma is located, played a role in self-pollination (e.g., Linnaeus, 1755; Sprengel, 1793), but since about the 1880s, it has generally been assumed that the stamen movement helps to precisely pack pollen on the tongues or faces of flies or bees (Kirchner, 1911; Knoll, 1956; Percival, 1965; Kugler, 1970; Lebuhn and Anderson, 1994). Kirchner (Kirchner, 1911) furthermore suggested that insects hit by a stamen would be encouraged to leave the flower, but soon would land on another flower to resume their nectaring. Rapid succession of brief visits to many flowers in Kirchner’s view should enhance cross-pollination and reduce nectar costs per pollen grain transport. A third possibility would be the above-mentioned visitor filtering if different kinds of insects were to react differently to forward-snapping of the stamens. This is the case, for example, in species of Opuntia, where forward-moving stamens make the pollen almost inaccessible to generalist bees and near-exclusively accessible to specialized bees that pollinate the flowers (Schlindwein and Wittmann, 1997).

In this study, we experimentally test these three hypotheses (filtering visitor species; metering out small numbers of pollen grains onto body parts likely to come in contact with conspecific stigmas; making pollinators leave more quickly) by immobilizing the stamens in the flowers of three species of Berberis and one species of Mahonia. Filament bending in Berberis relies on rapid changes in the calcium permeability of membranes (Lechowski and Białczyk, 1992), and we therefore explored treatments with calcium inhibitors and with alcohol. We discovered that immersion of flower pedicles in 75% alcohol for 35–45 min was effective at blocking the stamen movement. A test for possible effects of the alcohol treatment on foraging behaviors of the major floral visitors revealed no statistical effects. We then built experimental arrays with untreated and treated flowers in enclosed conditions to quantify pollen export and import from single visits of bees and flies and also used pollen staining to track pollen export distances from manipulated and control flowers.

Our experiments allowed us to address the following questions: (1) Are Berberis flowers with mobile stamens visited by the same types of insects and at the same rates as flowers with immobilized stamens? Different visitor types or visitation rates are expected under the filtering hypothesis, for instance, with flies reacting differently to touch-sensitive stamens than bees. (2) Do forward-snapping stamens make pollinators leave more quickly, reducing nectar costs per pollen grain transport? (3) Do flowers with touch-sensitive stamens export more pollen to more flowers and/or flowers further away? Lastly, we tested whether berberine, an alkaloid with antifeedant activity against herbivores found in Berberis leaves (Schmeller et al., 1997; Manosalva et al., 2019), is also present in Berberis pollen or nectar.

Results

Touch-sensitive stamen movements and experimental selfing and outcrossing

Flowers of Berberis and Mahonia have six petals and six stamens inserted between paired nectar glands (organ sizes are given in Supplementary file 1). Each pollen sac opens by a separate valve and contains about 610 ± 6 sticky yellow pollen grains that remain attached to the pollen sac (Figure 1, Figure 1—figure supplement 1A, Figure 1—figure supplement 2). When a flower visitor (or a pointed object) contacts the adaxial surface of a filament base, the respective stamen rapidly moves toward the flower center, placing pollen grains on the visitor’s tongue (Figure 1A and B, Figure 1—figure supplement 2E and F). The stamen movement takes 0.44 ± 0.02 s in Berberis julianae, 0.17 ± 0.02 s in B. jamesiana, and 0.23 ± 0.04 s in B. forrestii (Supplementary file 1). Within 1 min, the stamen moves back from the flower center, where the single style with its large stigma is located, to the petal, taking 227.70 ± 10.06 s, 110.37 ± 6.64 s, and 155.31 ± 14.07 s, respectively, to return its original position (Supplementary file 1). In Mahonia bealei, the stamen movement takes 0.09 ± 0.01 s (N = 10 flowers), and 3.46 ± 0.71 s later, the stamen starts moving back, taking 7.74 ± 1.96 s to return to its original position (Video 2).

In B. julianae, fruit set in open-pollinated flowers (80.0 ± 6.4%) was significantly higher than in bagged and self-fertilized flowers (27.5 ± 7.1%; Figure 2). However, seed set per fruit did not differ between manually self-pollinated and cross-pollinated flowers. Self-pollen receipt by stigmas of B. julianae after a single stamen movement (14 ± 3 grains, N = 16) was only 6% of the pollen receipt resulting from a single visit by the most common visitors, Apis cerana, which deposited between 230 and 260 grains (section ‘Effects of forward-snapping stamens on visitor behavior and pollination in the 22 field and under-enclosed conditions’) and roughly 1% of the pollen grains of a single anther with its two pollen sacs (ca. 1220 grains), indicating that intra-flower self-pollination mediated by the stamen movements plays a minor role in total pollen receipt.

Flower visitors and pollinators

At our study site, B. julianae was visited mainly by five insect species (Supplementary file 2), the bee A. cerana Fab., 1793 (Figure 1A), two anthophorid bees Anthophora waltoni Cockerell, 1910, and Habropoda sichuanensis Wu, 1986 (Figure 1B), and the syrphid flies Rhingia campestris Meigen, 1822, and Meliscaeva spec. In the field, it was not always possible to securely distinguish the anthophorid bees, although H. sichuanensis was clearly more frequent, and some of our results, for example, on visitation rates, therefore pool these species. These bees foraged for nectar, but not pollen, while the flies fed on both nectar and pollen (Figure 1C). Consistent with these feeding habits, pollen transfer efficiency of the bees was significantly higher than that of the flies (Supplementary file 2).

Tests for a possible confounding effect of the alcohol treatment on visitor behavior

We found no effect of any lingering alcohol scents (in stamen-immobilized flowers) on visitor behavior: Visitation rates of A. cerana to B. julianae (under enclosed conditions) did not differ between untreated flowers with mobile stamens (SM), flowers with immobilized stamens (SI) via alcohol immersion, and untreated flowers in a fixed position above alcohol, called SMA flowers (Wald χ² = 0.194, df = 2, p=0.908; Figure 3A). However, A. cerana stayed longer in SI compared to SM and SMA flowers (Wald χ² = 64.599, df = 2, p<0.001; Figure 3B), showing that it was the forward-snapping stamen that caused these bees to leave. Visitation rates of A. cerana to B. julianae (again under enclosed conditions) also did not differ among SM, SI, and filament-damaged (FD) flowers (Wald χ² = 0.44, df = 2, p=0.802; Figure 3C) because all three types of flowers offered the nectar sought by these bees. However, bees stayed less time in SM flowers than in flowers without stamen bending (SI and FD flowers) and equally long in SI and FD flowers (Figure 3D), showing that it was the stamen forward-snapping per se that caused visitors to move on.

Figure 3. — Stamens mobile (SM, controls), stamens immobilized (SI), natural flowers in a fixed position above alcohol (SMA), and flowers with their filaments damaged (FD) so that the stamens became immobile but retained their pollen sacs and the nectar glands to the right and left of each filament. The box plots indicate the median (mid lines), the interquartile range (boxes), and 1.5× the interquartile range (whiskers). Different lowercase letters indicate significant differences, and the numbers in brackets indicate sample sizes. Sample sizes (n) are given in brackets.

Effects of forward-snapping stamens on visitor behavior and pollination in the field and under enclosed conditions

In the field, when three or four SI flowers of B. julianae were inserted on racemes with the same number of SM flowers (Figure 1H), A. cerana stayed much longer on SI flowers than on SM flowers (15.46 ± 1.54 s vs. 3.63 ± 0.33 s, Wald χ² = 56.055, p<0.001 in 2020; 16.076 ± 1.515 s vs. 5.675 ± 0.382 s, Wald χ² = 68.421, p<0.001 in 2021). Despite the longer visits, fewer pollen grains were loaded onto A. cerana after a single visit to SI flowers than to SM flowers (716 ± 85 vs. 1223 ± 100 grains in 2020; 890 ± 73 vs. 1401 ± 134 grains in 2021; Wald χ² = 12.873, p<0.001 in 2020; Wald χ² = 10.511, p=0.001 in 2021), and the numbers of pollen grains deposited on stigmas by a single A. cerana visit also were much lower in SI flowers than in SM flowers (87 ± 10 vs. 230 ± 25 grains in 2020; 63 ± 8 vs. 260 ± 35 grains in 2021; Wald χ² = 26.847, p<0.001 in 2020; Wald χ² = 40.042, p<0.001 in 2021; Figure 5, Figure 5—figure supplement 1). Pollen transfer efficiency by A. cerana was therefore reduced in SI compared to SM flowers, and this was significant in 2021 but not 2020 (0.084 ± 0.016 vs. 0.303 ± 0.102; Wald χ² = 4.505, p=0.034 in 2021; 0.205 ± 0.024 vs. 0.246 ± 0.091; Wald χ² = 0.188, p=0.665 in 2020; Figure 5—figure supplement 1I and J).

When the experiment was repeated under enclosed condition (using five SI and five SM flowers), the bees and syrphid flies all stayed longer on SI than on SM flowers (33.7 ± 4.2 s vs. 16.0 ± 1.7 s; Wald χ² = 30.106, p<0.001; Figure 4E′–T). All visitor species exploited more nectar (Figure 4M′–P) and touched more stamens in SI than in SM flowers (Figure 4I′–L). The pollen transfer efficiency of the bees was higher than that of the flies (Wald χ² = 13.319, df = 3, p=0.004, N = 80). In SI flowers, A. cerana was loaded with fewer grains than in SM flowers (just as under outdoor conditions; above), while the anthophorid bees and the flies under enclosed conditions removed more grains from SI flowers than from SM flowers (Figure 4Q′–T).

Figure 4. — The major pollinator, *Apis cerana*, was studied in 2020 and 2021 under open pollination (far left) and enclosed conditions (set-up shown in Figure 1—figure supplement 1 and Video 3), whereas the anthophorid bee pollinators and the two flies (*Meliscaeva* spec. and *Rhingia campestris*) were compared under enclosed conditions in 2021. Different lowercase letters above error bars indicate significant differences between control (stamen mobile [SM]) and alcohol-treated (stamen immobilized [SI]) flowers. (**A′–D**) Visitation rates of four visitor species, showing that *A. cerana* visited control flowers more frequently than SI flowers under open pollination (A′), but no visitor species discriminated between SI and SM flowers under enclosed conditions (**A–D**). All visitor species spent more time (**E′–H**) and touched more stamens (**I′–L**) in SI flowers than in control SM flowers. Visitors removed more nectar from SI flowers, resulting in less nectar remaining per flower (**M′–P**). Pollen removal by *A. cerana* was lower from SI than from SM flowers (Q′, Q), but higher in the other three visitor species (**R–T**). Compared to SM flowers, pollen transfer efficiency was significantly decreased in SI flowers (U′, **U, V, X**), although it did not differ in *Meliscaeva* spec. (W). Sample sizes (n) are given in brackets.

Effects of mobile stamens on pollen receipt in experimental visits

After experimental single visits, both A. cerana (Figure 5A, Wald χ² = 142.565, p<0.001) and the anthophorids (Figure 5B, Wald χ² = 14.236, p<0.001) carried fewer pollen grains on their tongues after visiting SI flowers compared to SM flowers.

Figure 5—figure supplement 1. — Numbers of pollen grains placed on bees’ tongues during a single visit were significantly higher when stamens were mobile (SM) than when stamens were experimentally immobilized (SI) (**A, B**). Numbers of pollen grains deposited on the stigma of the second-visited flower (pollen recipient) during single visits by *A. cerana* (C) and the anthophorids (D) in three trials with the sequence being SM + SM flowers; SM + SI flowers; and SI + SI flowers. The box plots indicate the median (mid lines), the interquartile range (boxes), and 1.5× the interquartile range (whiskers). Different lowercase letters indicate significant differences among three trials.

In three trials in which we held anesthetized bees between forceps and simulated visits to SM and SI flowers in different sequences (‘Materials and methods’), the number of pollen grains deposited on stigmas by A. cerana (mean ± SE, Figure 5C) and the anthophorids (mean ± SE, Figure 5D) in trial 1 (SM + SM flower) was significantly higher than in trials 2 (SM flower + SI flower) and 3 (SI + SI flowers; Wald χ² = 118.887, p<0.001 vs. Wald χ² = 69.274, p<0.001, respectively). Moreover, pollen receipt by A. cerana and the anthophorids was significantly higher (p<0.001) in trial 2 (SM + SI) than in trial 3 (SI +SI), indicating that the stamens precisely place pollen grains on tongues, which then deposit them on stigmas during the insect’s next flower visit.

Effects of mobile stamens on pollen export and receipt in the field

Pollen export of SM and SI flowers was quantified with pollen-tracking experiments in which pollen of flowers of B. julianae and B. jamesiana was stained in situ with either eosin or aniline blue, and stigmas of all flowers in the vicinity (about 25–100 cm from the source plant) were checked for stained grains.

Of over 700 flowers of B. julianae whose stigmas we checked, 44 of 772 flowers received pollen from SM flowers and 14 of 733 flowers from SI flowers (Supplementary file 3), indicating that flowers with mobile stamens donated pollen to about 3.1× more flowers (44/772 = 0.057 vs. 14/733 = 0.019, G = 15.341, p<0.001). The mean number of pollen grains deposited per stigma in these trials was also higher from SM than SI flowers (0.16 ± 0.03 vs. 0.03 ± 0.01; Wald χ² = 76.536, p<0.001). All four runs of this experiment showed a consistent pattern: more pollen grains from SM flowers were delivered to more flowers (Figure 6, Figure 6—figure supplement 1).

Figure 6. — Mean pollen number and SEs (numbers of flowers with stained pollen deposition/total number of sampled flowers of pollen recipients) are given for treated and control flowers in each of four trials (**A–D**). Note that only 15 flowers are shown, although each pollen-tracking test sampled over 100 flowers to examine the effect of stamen movements on pollen dispersal; for example, of 260 pollen-recipient flowers, only 4 flowers received pollen grains from stained stamens-immobilized (SI) flowers, that is, (4/260) in (A).

Within <50 cm from the dyed pollen source, 25 of 338 flowers received pollen from SM flowers, while only 10 of 395 flowers received pollen from SI flowers (G = 9.645, p=0.0019, Supplementary file 3). At distances of 50–100 cm, 19 of 434 flowers received pollen from SM flowers, while only 4 of 338 flowers received pollen from SI flowers (G = 7.439, p=0.0064, Supplementary file 3), and at distances >100 cm, only pollen from SM flowers was detected on stigmas (on 6 of 222 inspected flowers).

When we conducted the same experiment in B. jamesiana (Figure 6—figure supplement 1), 75 of 400 nearby flowers received pollen from SM flowers and 40 of 400 flowers from SI flowers, again indicating that mobile stamens were likely to donate pollen to more flowers (75/400 vs. 40/400, G = 12.612, p<0.001). The mean number of pollen grains deposited per stigma was also higher in SM than SI flowers (0.4 ± 0.05 vs. 0.2 ± 0.04; Wald χ² = 22.95, p<0.001).

Within <25 cm from the dyed pollen source, 13 of 60 flowers received pollen from SM flowers, while 9 of 46 flowers received pollen from SI flowers (G = 0.07, p=0.791). At distances >25 cm, 7 of 60 flowers received pollen from SM donors, while only 1 of 56 flowers received pollen from SI flowers (G = 4.961, p=0.026, Supplementary file 4).

Berberine content in B. julianae leaves, petals, pollen, and nectar

High-performance liquid chromatography (HPLC) of the berberine concentrations in B. julianae leaves, petals, pollen, and nectar indicated high berberine concentrations in leaves, petals, and pollen, while no berberine was detectable in the nectar (Supplementary file 5). That bees can taste the berberine in the pollen is suggested from the observation that individuals of all bee species used their front legs to remove pollen grains that stuck to their tongues.

Discussion

Insect-induced movements of flower parts, including styles or stamens, have fascinated botanists since Linnaeus (1755) but their adaptive significance has been difficult to investigate because immobilization under field conditions has been difficult. As far as we are aware, this is the first study to directly study visitor behavior and pollen import/export in flowers with experimentally immobilized stamens, using our discovery that an alcohol solution blocks stamen movement in Berberis and Mahonia flowers. We developed a simple protocol of immersion of pedicels in alcohol and then carefully inserted flowers with immobilized stamens in racemes with the same number of natural flowers (directly in the field) or exposed them to visitors in experimental arrays (under enclosed conditions). This allowed us to test three hypotheses explaining the adaptive value of touch-sensitive stamens in Berberidaceae: visitor filtering; making pollinators leave more quickly, thus reducing nectar costs per pollen grain movement; and precise metering out of few pollen grains per visitor, enhancing pollen export. We found support for the last two, but not for the filtering hypothesis, since flies and bees reacted similarly to stamen bending. In the following, we discuss these results in more detail.

To maximize pollen dispersal, the number of grains removed by each visitor should be limited so as to heighten the probability of pollen reaching different conspecific stigmas (Harder and Thomson, 1989), and where possible, visitors should be ‘paid’ by replenishable nectar rather than pollen (Westerkamp, 1996). In Berberis, such metering out of pollen is achieved by the individual stamens only bending forward once their adaxial filament base is touched by an insect’s tongue probing the nearby nectaries. Further protection of pollen grains from exploitation by pollen thieves or inefficient vectors is sometimes achieved by chemical defense (Palmer‐Young et al., 2019; Wang et al., 2019), and we therefore examined whether the pollen grains of Berberis contain berberine, an alkaloid with antifeedant activity against herbivores and pests (Schmeller et al., 1997; Manosalva et al., 2019). This is indeed the case (Supplementary file 5), and in B. julianae, we observed bees cleaning off and discarding pollen grains that stuck to their tongues with their front legs. The syrphid flies, however, fed on the pollen despite its berberine content. There is therefore no support for a chemical defense against pollen thieves (here pollen-feeding flies) and some support for bees being discouraged from collecting Berberis pollen as food for their larvae. Importantly, the syrphids visiting B. julianae are only conditional pollen thieves (Hargreaves et al., 2009) since the species is self-compatible and even self-pollen deposited by flies or by stamens hitting the flower’s own stigma contributes to reproductive insurance.

The quantitative effect of stamen bending on the duration of bee visits per flower was large. Thus, in stamen-bending flowers, the bees typically stayed for five seconds and triggered 2–3 stamens per flower per visit (Figure 4), a similar number as in Berberis thunbergii in North America, where the main visitors also are medium-size bees of in the genus Apis and the family Anthophoridae (Lebuhn and Anderson, 1994). In stamen-immobilized flowers, bees stayed about 3× longer (on average 14.37 ± 1.53 s) and flies about 2× longer (on average 54.38 ± 7.53 s), and both visitor types therefore removed more nectar. The impact of longer stays on pollen removal, however, differed between the most common visitor, A. cerana, and the other three visitors: When immobilized stamens no longer smeared pollen grains on its tongue or face, A. cerana carried away fewer grains, while the pollen-feeding syrphid flies and the two anthophorid bees removed more grains, in the case of the flies because they ate more pollen and in the case of the anthophorids because they passively touched more open anthers, the longer they stayed. The quicker leaving of flowers not only reduced nectar costs/pollen grain transported but also greatly increased male reproductive success by dispensing more pollen to more recipients. This is evident from pollen dispersal distances in SM and SI flowers of B. julianae (Figure 6, Supplementary file 3) and B. jamesiana (Figure 6—figure supplement 1, Supplementary file 4), and from the proportions of pollen recipients that were reduced by 62.03% [=1 – (14/733 ÷ 50/994)] and 41.18% [=1–{(9+1)/(46+56) ÷ (13+7)/(60+60)}] in the two species.

While we found no support for touch-sensitive stamens filtering floral visitor types – all visitors left flowers more quickly after being hit by stamens– other studies on species with stamens triggered by flower visitors have found strong support for touch-sensitive stamens filtering out inefficient pollinators. Thus, in species of Opuntia, the stamens touched by bees move toward the flower center, creating a dense layer that effectively excludes generalist bees, while three specialized bee species are able to access the pollen (Schlindwein and Wittmann, 1997). And in Meliosma tenuis, only nectar-seeking bumblebee drones are able to trigger the stamens and are then loaded with pollen (which males do not collect as larval food), while other insects are unable to access the pollen (Wong Sato and Kato, 2018).

Recent reviews of the adaptive significance of the movement of floral parts (including both styles and stamens) have distinguished four types of stamen movement: (1) slow movement triggered by a visitor, (2) quick or even explosive movement (sometimes via a catapult mechanism) triggered by a visitor, (3) simultaneous slow movement occurring without triggering by a visitor, and (4) a ‘cascade’ or staggered movement in which one stamen moves after the other, regardless of triggering (Ren, 2010; Ruan and da Silva, 2011; Armbruster et al., 2014). The Berberidaceae, a family of some 700 species, exhibit aspects of two of these categories, namely, the staggered movement and the quick triggered movement, although not involving the sudden release of a built-up tension as, for instance, in Kalmia (Switzer et al., 2018) in which each filament is arched backward in a petal pocket. Experimental work on the costs and benefits all these types of stamen movements, which occur in hundreds of flowering plants, is still in its infancy, however, partly because it is so difficult to manipulate floral movements under field conditions.

Conclusion

Even though botanists have speculated about the adaptive value of the visitor-triggered forcefully forward-snapping stamens of Berberis since 1755 (Linnaeus, 1755), this is the first experimental investigation of how this trait impacts the flowers’ pollen export and receipt. Our results demonstrate surprisingly large effects of stamen bending on pollen export (involving both quantity and distance) and nectaring times (involving lower nectar costs per pollen grain transfer) and reveal another mechanism by which plants meter out their pollen.

Materials and methods

Plant and insect study species

During each March between 2019 and 2022, we studied a natural population of B. julianae C.K.Schneider in a field located at 29°52′26″N, 105°30′32″E, 427 m above sea level, about 50 km southeast of Anyue County, Sichuan Province, China. Experiments to evaluate the effect of stamen movements on pollen dispersal were also carried out in a natural population of B. jamesiana Forrest & W.W.Smith at Shangri-La Alpine Botanical Garden (27°54′05″N, 99°38′17″E, 3300–3350 m above sea level), Yunnan Province, Southwestern China. We also studied planted populations of B. forrestii Ahrendt at the Shangri-La field station and of M. bealei (Fortune) Carrière in the Wuhan Botanical Garden (30°33′2″N, 114°25′48″E, 23 m above sea level) in Hubei Province to test the effects of the alcohol treatment on stamen mobility. Our Berberis and Mahonia taxonomy follows the Flora of China (Ying et al., 2011). Herbarium vouchers of each species have been deposited in the herbarium of Central China Normal University (CCNU). All species are hermaphroditic perennial shrubs with clusters of 9–25 yellow flowers produced between March and May, depending on species. Individual flowers last for 3–5 days, and each anther dehisces upward by two valves exposing the pollen grains (Figure 1). The bottle-shaped pistils have one ovary containing 2–4 ovules with a discoid stigma with a peripheral ring of papillae (Figure 1E).

Insect visitors were observed, in some cases filmed, captured with insect nets in the field, and preserved for later identification by insect taxonomists.

Alcohol as an inhibitor of the stamen movement, and tests for confounding the effects of the alcohol treatment on visitor behavior

When we immersed floral pedicels in a solution of 75% alcohol for 40 min, all stamen movement was blocked (Videos 1 and 3 of B. julianae and Video 2 of M. bealei). To test whether any lingering alcohol scent affected visitor behavior in alcohol-treated flowers, we set up arrays with different types of flowers as follows. We first bagged >20 flowers on different individuals of B. julianae before they opened. Once open, 18 flowers were gently cut off and subjected to one of the three treatments: (1) stamen-mobile (SM) flowers: six flowers without any treatment; (2) stamen-immobilized (SI) flowers: six flowers whose pedicels (ca. 10 mm long) were immersed in 75% alcohol for about 40 min; and (3) six natural flowers in a fixed position above alcohol (SMA flowers). For this, the pedicels of freshly opened flowers were inserted into 30-mm-long Eppendorf microcentrifuge tubes that contained 10 μL alcohol, such that they did not touch the alcohol (Figure 1—figure supplement 1A). All 18 flowers were then fixed in position by inserting them into small holes on the surface of a paper box covered by a clean glass cup (as shown in Figure 1—figure supplement 1). We then placed freshly caught Berberis visitors inside the glass cup so they could interact with the enclosed floral arrays and recorded visitation rates (visits per flower per 10 min) and handling times.

Video 1. Stamens of Berberis julianae become touch-insensitive after the flower pedicels had been immersed in 75% alcohol for 35 min.

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Video 2. Stamens of Mahonia bealei become touch-insensitive after the flower pedicels had been immersed in 75% alcohol for 30 min.

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Video 3. Under enclosed conditions (Figure 1—figure supplement 1D), individual syrphid flies (Meliscaeva spec.) took up nectar from a Berberis julianae flower with immobilized stamens for much longer, giving them time to touch four stamens, while leaving more quickly and touching only two stamens in a control flower with mobile stamens.

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To further examine the possible effects of the alcohol treatment versus the loss of stamen movements on the duration of insect visits, we set up additional floral arrays consisting of three types of flowers: six SM flowers, six alcohol-treated SI flowers, and six FD flowers, the latter being flowers in which all six filaments were damaged with clean forceps so that the stamens could not move but petal nectaries and the anther sacs were still there (Figure 1—figure supplement 1C). The experimental procedure for these arrays was the same as above (Figure 1—figure supplement 1B).

Fruit and seed set after self-pollination vs. cross-pollination, and contribution of stamen forward-snapping to selfing

To test whether B. julianae is self-compatible and whether fruit or seed production are pollinator-limited, in 2020 and 2021, 80 flowers from 12 individuals were subjected to the following four pollination treatments. (1) Control: 20 randomly chosen flowers from 12 individuals were open pollinated, while the remaining 60 flowers were bagged and subjected to one of three treatments: (a) automatic self-pollination: flowers not manipulated; (b) self-pollination: flowers hand-pollinated with pollen mixtures collected from flowers of the same individual; (c) cross-pollination: flowers hand-pollinated with pollen mixtures from multiple flowers of individuals at least 20 m away. Flowers were then bagged with mesh until the petals dropped 1 week later. Fruits were harvested 3 months later and seeds and undeveloped ovules per fruit counted. Fruit set was calculated as fruit number divided by flower number in each treatment. Seed set was calculated as seed number per fruit divided by total number of seeds and undeveloped ovules. Aborted fruits were not included.

To examine intra-flower selfing induced by the stamen movement, we counted pollen grains deposited per stigma under a light microscope in 16 bagged flowers in which we had triggered one stamen by a needle.

Effect of stamen bending on visitor behavior and pollen export and import

Flowers of B. julianae were visited by several species of bees and two species of syrphid flies (‘Results’). To examine the effects of stamen bending on foraging behavior and pollen transfer, floral visitors were allowed to interact with the above-described flower arrays (section ‘Alcohol as an inhibitor of the stamen movement, and tests for confounding the effects of the alcohol treatment on visitor behavior’) in the field (Figure 1) and under enclosed conditions (Figure 1—figure supplement 1D). Under enclosed conditions, we compared visitation rates, handling times, numbers of stamens touched, nectar volume remaining, pollen removal, and pollen receipt on the stigma after single visits of the four species of visitors. We also quantified pollen transfer efficiency after single visits by harvesting flowers that had been visited a single time in the field and then counting pollen grains on their stigmas as well as pollen grains remaining in their stamens. To quantify pollen loads placed on insect bodies and stigmas of next-visited conspecific flowers, we held captured and anesthetized bees between forceps and made their tongues contact the filament bases in SI or SM flowers. Using this method, we carried out three trials with SM and SI flowers: trial 1, SM + SM; trial 2, SM + SI; and trial 3, SI + SI flowers.

Pollen-tracking experiments to quantify the effects of stamen forward-snapping on pollen export distances

To quantify pollen export from SM and SI flowers, we conducted four pollen-tracking experiments each in B. julianae in March 2022 and B. jamesiana in May 2021. Each trial was conducted on a sunny day and involved 60 flowers from 3 to 5 individuals whose pollen was stained as the pollen donor: 30 flowers were alcohol-treated (SI flowers) and the remaining 30 flowers were SM flowers. The manipulated flowers were carefully inserted among natural flowers on densely blooming racemes. The stigmas of >100 flowers (126–260) on the same and on different individuals at varying distances from the donor were then examined for dyed pollen grains, these were counted, and the straight-line distance from donor to recipient recorded with a meter rule.

Statistical analyses

Visitation rates of visitor species in the field (not normally distributed) were examined with nonparametric Kruskal–Wallis tests. To compare visitor behavior on SM, SMA, FD, and SI flowers, we performed a generalized linear model (GLM) analysis with normal distributions and identity-link function to test for differences in visitation rates, handling time, and pollen transfer efficiency (log10-transformed) of the four main visitor species. Nectar volumes remaining after different visitors had visited were examined with a nonparametric Kruskal–Wallis test. To compare the number of stamens touched in different flowers, pollen removal, and pollen deposition by each visitor species, and pollen export and import, we performed GLM analyses with a Poisson distributions and loglinear-link function. Floral traits among species were compared under a GLM with a normal distribution and identity-link function, while for numbers of pollen ovules per flower we used a Poisson distribution and loglinear-link function (Supplementary file 1). Data of pistil height and stamen length were log10-transformed to achieve normal distribution. We used the G-test of independence to test whether stained pollen from SM or SI donors differs in the distances to which it is dispersed to (McDonald, 2014). A GLM with binomial distribution and logistic-link function was used to detect the effects of the selfing and outcrossing on fruit set and seed set (with fruit/seed number as event variable, total treated flower/ovule number as trial variable, and different treatments as factors). The GLM analyses were performed in SPSS 19.0 (IBM, Armonk, NY).

Acknowledgements

We thank lab members Q-M Quan, X-W Lv, and Z-X Tian for field assistance and Z-Y Tong, and Y-Z Xiong for methodological and statistical advice; Dr. Huan-Li Xu, Department of Entomology, China Agricultural University, Beijing, for the identification of flies and bees; Dr. Antti Haarto, Zoological Museum, University of Turku, Finland, for the identification of Meliscaeva; Dr. Michael Orr, Institute of Zoology, Chinese Academy of Sciences, Beijing, for the identification of Habropoda cf. sichuanensis; Dr. Chih-Chieh Yu for confirming our plant identifications; Z-D Fang and staff of Shangri-La Botanical Garden for logistical support; Sarah Corbet, Steven Johnson, and Nathan Muchhala for providing helpful suggestions on early versions of this manuscript; and the reviewers Bernhard Schmid, Zong-Xin Ren, and Felipe Yon for their constructive comments. This work was supported by the National Natural Science Foundation of China (grant nos. 31730012 and 32030071) and Fundamental Research Funds for the Central Universities (no. CCNU22LJ003) to S-QH.

Appendix 1

Measurements of floral traits in Berberis and Mahonia species

Flower size, length of the pedicel, and other flower traits were measured with a digital caliper to 0.01 mm (Supplementary file 1). To estimate nectar volume per flower of B. julianae, clean 10 µL microliter syringes (Agilent Technologies Inc, USA) were used to extract nectar drops from bagged flowers. To count pollen grains and ovules per flower, we randomly collected one of six anthers from virgin flowers that had been bagged as buds with fine-mesh cotton bags to exclude visitors. Each selected anther was placed on a microscope slide and then squashed under a coverslip. All pollen grains were counted under a microscope, and the number in one anther was multiplied by six to obtain the pollen grain number per flower. Meanwhile, ovules of sampled flowers were also counted. Sample sizes are shown in Supplementary file 1.

Testing protocols for best alcohol treatments to block the stamen movement

To find the minimum time required to completely block stamen movements in each of the three species of Berberis, we checked the response of stamen movements in a time series of alcohol-treated flowers. Pedicels were gently cut off and the bases (about 10 mm long) immersed in 75% alcohol. Every 5 min, 8–10 flowers were checked by touching the filaments of each flower with a dissecting needle to identify whether the stamens remain mobile. For B. julianae, we tested 64 flowers, for B. jamesiana 100, and for B. forrestii 80.

Floral visitors and effect of stamen bending on their behavior and pollen export and import

Visitation rates (visits/flower/hour) of the different visitors to B. julianae were obtained on sunny days in March 2019, 2020, and 2021. Visitor were observed on fully flowering shrubs with over 100 open flowers, and each observation period lasted 1 hr, during 9:00–12:00 and 12:30–16:30. The sex and foraging behavior of each visitor were recorded, such a feeding on nectar or pollen or groomed pollen grains into pollen loads. Total observation times in the 3 years are shown in Supplementary file 2.

To see whether floral visitors discriminated between SI and SM flowers, we compared visitation rates of the Asian honeybee A. cerana under open pollination and under enclosed conditions. The other insect species were only studied under enclosed conditions (illustrated in Figure 1—figure supplement 1).

In March 2020, 72 flowers on different individuals of B. julianae were randomly bagged with mesh nets, before they opened. Also, 12 SI and 12 SM flowers were observed each day. When flowers were beginning to open, we gently cut off 36 flowers (12 flowers × 3 days) at the base of the pedicels and immersed the pedicels in 75% alcohol. All stamens in each alcohol-treated flower became touch-insensitive in 40 min. These SI flowers were carefully inserted into racemes of flowering individuals with the other 36 SM flowers on these racemes as controls, allowing the bees to visit in open pollination conditions (Figure 1D and H). The number of flowers visited per hour was recorded during 09:00–12:00 and 13:00–17:00 for 3 days. Visitation rates were calculated as the number of flowers visited per hour divided by the number of observed flowers, that is, visits per flower per hour.

In March 2021, a total of 160 floral buds from five individuals were randomly bagged. As above, 80 newly opened flowers were alcohol-treated and the remaining 80 flowers were free of alcohol as SM flowers. On each observation day, we set up four arrays each having four SM and four SI flowers. The visitation rates were recorded during 09:30–12:30 and 13:00–17:00 for 5 days each with 16 SI and 16 SM flowers (Figure 5—figure supplement 1A-B).

To compare the handling time, pollen removal, and pollen transfer efficiency of the pollinator A. cerana in SM and SI flowers under open conditions, 100 flowers from different individuals of B. julianae were randomly bagged with mesh before the flowers opened in March 2020 and 2021. When the flowers were newly opened, 12 flowers per day were alcohol-treated for 5 days per year with fine weather. Four of the SI flowers were carefully inserted into each of three racemes with buds, but no open flowers (4 flowers × 3 racemes × 5 days = 60), and four bagged SM flowers from two racemes were uncovered per day (4 flowers × 2 racemes × 5 days = 40) for A. cerana visits. In the five arrays, each of four flowers per day, we recorded handling times of A. cerana during each visit to one flower. To estimate pollen transfer efficiency, 20 SI and 20 SM flowers visited once by A. cerana were harvested immediately in the field. Six anthers and the stigma of each flower were collected. We counted pollen grains remaining within anthers per flower and deposited per stigma under a light microscope. Pollen removal per flower was calculated as the mean number of pollen grains (see ‘Results’) minus the remaining grains. Pollen transfer efficiency was calculated as pollen deposition divided by pollen removal. In both years, we obtained 20 pairs of pollen removal and deposition data for SM flowers, and 20 pairs for SI flowers. In March 2021, we also compared the number of stamens touched and the remaining nectar volume left by A. cerana in SM and SI flowers that it had it visited just once. As visitor switched to collect nectar from another nectary, their bodies turned. Therefore, counting the visitor’s body turns allowed us to record the number of stamens touched by a visitor in SI flowers (Video 3).

To estimate nectar collection by different visitors, we measured the remaining nectar volume within each SM and SI flower after one visit using a clean 10 µL microliter syringe (Agilent Technologies Inc).

To compare the foraging behavior of bees and flies on SM and SI flowers under enclosed conditions (Figure 1—figure supplement 1), we set up floral arrays each consisting of five SM and five SI flowers. The flowers were fixed by inserting the pedicels into small holes on the surface of a paper box covered by a clear glass cover. We then allowed individual of the different insect species to freely interact with the floral array. We recorded the visitation rates (visits per flower per 10 min) and handling time of the visitor and the number of stamens touched by the visitor in each flower, measured the nectar volume left by the visitor, and assessed pollen removal and pollen deposition after one visit to calculate pollen transfer efficiency. It took four trials of ‘interviews’ (cafeteria experiments) over 2 days to yield 20 once-visited flowers for each insect species. Four individuals of each insect species were captured in the field and released within 2 hr after their visits to the SM and SI flowers.

Pollen export/import after single visits

To estimate the effects of the stamen mobility on pollen placement on the pollinator and on stigmas, we simulated bee visits by to SI and SM flowers. In March 2021, we bagged 320+ flower buds from different individuals of B. julianae. When the flowers opened, we held anesthetized bees between forceps so that they probed the flower, pollen grains placed on the bee’s tongue (and occasionally on the head) were pasted onto a piece of tape. The tape was attached to a slide and pollen grains placed on the bee’s tongue in 20 SI and 20 SM flowers were then counted under a light microscope (Figure 5A and B). Using this method, we also carried out three trials with simulated visits to flowers in specific sequence: Trial 1, SM + SM flowers; trial 2, SM + SI; and trial 3, SI + SI flowers. Each trial was repeated 20 times. After each trial, the stigma of the second-visited flower (the pollen recipient) was collected to count the pollen grains deposited on its stigma (Figure 5C and D).

Pollen-tracking experiments to quantify the effects of stamen forward-snapping on pollen export distances

To examine the effects of stamen movements on the fate of pollen, we conducted four trials of pollen-tracking experiments in a transplanted population of B. jamesiana at Shangri-La Alpine Botanical Garden, where the interference of stained pollen with the sexual reproduction of wild plants could be minimal. Trials were conducted on sunny days and involved 60 flowers from 3 to 5 individuals whose pollen was stained as pollen donors: 30 flowers were alcohol-treated (SI flowers) and the remaining 30 flowers were used as SM flowers. Pollen grains in SM or SI flowers were stained with eosin (stained red) or aniline blue (blue). The two dyes were alternately used between SM and SI flowers in four trials. The dyes dried within 5–10 min, and all pollen-stained flowers were taken to the field and carefully inserted into racemes of two flowering individuals along the roadside for pollinators to visit. To track pollen flow, the two clusters containing either 30 SM or SI flowers were arranged separately (about 100 cm apart) on the flowering branches. Flowers of each cluster were within a 40 cm × 40 cm square on four erect racemes. Previous studies indicated that pollen flow mediated by generalist insects usually occurs within meters, with a highly leptokurtic distribution (Williams, 2001). One day (24 hr) later, given that A. cerana visits were infrequent to B. jamesiana in May 2021, stigmas of 100 flowers from nearby racemes were collected. Dyed pollen grains deposited on each stigma were counted under a stereomicroscope.

To compare the distance of pollen dispersal from SM and SI flowers, we conducted two trials of pollen-tracking experiments to compare distance of pollen dispersal between SM and SI flowers of B. jamesiana. Each trial was the same as above. At 17:30 on the day of the experiment, stigmas of all flowers from nearby racemes were collected and we noted the straight-line distance within 25 cm and over 25 cm from the racemes with the sampled flowers to the racemes with pollen-stained SM or SI flowers. Dyed pollen grains deposited on each stigma were counted under a stereomicroscope.

Measurement of chemical defense in pollen and nectar

To examine whether berberine is present in Berberis pollen and nectar, we collected leaves, petals, pollen, and nectar from 10 plants of B. julianae. Leaves, petals, and pollen grains were dried using an oven at 65°C, while the nectar was stored at –20°C before chromatographic analysis. The berberine content of the different samples was analyzed using HPLC. To extract berberine from leaves, a 0.1 g leaf sample was weighed with a balance (Sartorius BAS124S) and transferred to a 2 mL microcentrifuge tube. A steel bead was added to the tube. The leaves were ground using a tissue homogenizer (Tissuelyser, QIAGEN) at 30 Hz for 10 min, and then the leaf sample was transferred to a vial. Enzymolysis of the leaf sample was conducted by adding 2 mL pure water, 1 μL dilute sulfuric acid, and 6 mg cellulose at 50°C in a magnetic stirrer for 3 hr. The leaf sample was extracted for 2.5 hr in 5 mL pure water, and then transferred to a 5 mL microcentrifuge tube. Following centrifugation at 8000 rpm for 10 min (Centrifuge 5430R, Eppendorf), the supernatant was transferred to a vial. The volume of the sample was recorded. Berberine in petals, nectar, and pollen grains was measured similarly.

Components were separated using an Acquity HPLC BEH C18 column (50 × 2.1 mm, 1.7 μm) (Waters, Milford, MA) set at 30°C, and the injection volume was 20 μL. All aspects of system operation and data acquisition were controlled by software (Agligent 1100 series) at the Center of Analysis and Test of Wuhan University, Wuhan, China. The mobile phase was acetonitrile–0.3% phosphoric acid–pure water (35:5:60); flow rate: 1.0 mL/min; determination wavelength: 346 nm; the detection signal was diode array detector (DAD). The berberine concentrations in the different samples were compared using a GLM with normal distribution and identity-link function.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Shuang-Quan Huang, Email: hsq@ccnu.edu.cn.

Bernhard Schmid, University of Zurich, Switzerland.

Jürgen Kleine-Vehn, University of Freiburg, Germany.

Funding Information

This paper was supported by the following grants:

National Natural Science Foundation of China grants no. 31730012 and 32030071 to Shuang-Quan Huang.
Fundamental Research Funds for the Central Universities no. CCNU22LJ003 to Shuang-Quan Huang.

Additional information

Competing interests

No competing interests declared.

Author contributions

Resources, Formal analysis, Investigation, Methodology, Writing – original draft.

Methodology.

Writing – original draft, Writing – review and editing.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Writing – original draft, Project administration, Writing – review and editing.

Additional files

Supplementary file 1. Floral traits (mean ± SE) and duration of stamen movements in Berberis jamesiana, B. julianae, B. forrestii, and Mahonia bealei.

elife-81449-supp1.docx^{(15.7KB, docx)}

Supplementary file 2. Foraging behaviors of two types of major insect visitors to Berberis julianae.

Behaviors of the bees and flies visiting Berberis julianae, focusing on visitation rates (visits per flower per hour, mean ± SE), visits per flower, handling time, number of stamens touched, nectar volume remaining per flower, pollen grains removed, pollen grains deposited, and pollen transfer efficiency.

elife-81449-supp2.docx^{(16.4KB, docx)}

Supplementary file 3. G-test of independence confirming that Berberis julianae flowers with mobile stamens (SM flowers) donated pollen to more recipient flowers at three distance classes than did flowers with experimentally immobilized stamens (SI flowers).

elife-81449-supp3.docx^{(13.2KB, docx)}

Supplementary file 4. G-test of independence confirming that Berberis jamesiana flowers with mobile stamens (SM flowers) donated pollen to more recipient flowers at two distance classes than did flowers with experimentally immobilized stamens (SI flowers).

elife-81449-supp4.docx^{(12.8KB, docx)}

Supplementary file 5. Concentrations of berberine (mean ± SE) in different tissues of Berberis julianae under generalized linear models.

elife-81449-supp5.docx^{(13KB, docx)}

MDAR checklist

elife-81449-mdarchecklist1.docx^{(99.2KB, docx)}

Data availability

All data are shown in the main text and the tables at the end of the main text.

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eLife. doi: 10.7554/eLife.81449.sa0

Editor's evaluation

Bernhard Schmid ¹

With a series of manipulative experiments using four plant species with stamens that can snap toward the stigma if touched at the base, the authors provide compelling evidence that pollinators stay longer yet export less pollen to recipient flowers when stamens are immobilized by alcohol application. This is a landmark study on the functional consequences and adaptive significance of a phenomenon scattered throughout the angiosperm clade.

eLife. doi: 10.7554/eLife.81449.sa1

Decision letter

Editor: Bernhard Schmid¹

Reviewed by: Bernhard Schmid², Zong-Xin Ren³, Felipe Yon⁴

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "Mobile stamens enhance pollen dispersal by scaring floral visitors away" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, including Bernhard Schmid as Reviewing Editor and Reviewer #1, and the evaluation has been overseen Jürgen Kleine-Vehn as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Zong-Xin Ren (Reviewer #3); Felipe Yon (Reviewer #4).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The different experiments need to be described in more detail both with regard to the design, procedure, measurements, and analyses. Provide more biological description of the receptivity, fertility, perception, day-cycle, and snapping capacity of the flowers over the 5 days that they are assumed to be opened for all species, including potential auto-deposition by spontaneous snapping.

2) Of the four hypotheses the first two have been tested with good experimental designs and statistical power (pollen export from mobile vs. immobile stamens). However, this was not the case for the other two (pollinator filtering, presence or effect of berberine in pollen on pollinators). This should be acknowledged and discussed throughout the manuscript.

3) Check that the entomology is correct, including species identity and natural history. Also, be careful regarding the interpretation of pollen removal as indicative of berberine content in pollen.

4) The Discussion should be improved by better synthesizing the different experiments into a general narrative and referring more extensively to general theory and other work on pollination aspects considered in this paper. Also, point out limitations and what would have to be done next to get a more comprehensive and mechanistic understanding of the function of mobile stamens (including pollinator behavior and niche separation as well as pollination results such as increased outcrossing rate or distance).

Reviewer #1 (Recommendations for the authors):

Besides the main results whether mobile stamens could "filter" visits according to pollinator species identity could not be confirmed and whether berberine affects pollinator behavior could only weakly be inferred from observation of pollen removal from bee tongues. It also remains to be shown whether mobile stamens additionally have a function in ensuring auto-deposition of pollen on stigmas for selfing and whether they increase seed production and offspring fitness of the plant.

While the experiments carried out are fine as far as they go, they are somewhat haphazardly planned using different species and places to test different partial questions relevant to the whole story. There is also some intrinsic confounding between different possible functions of mobile stamens, two mentioned by the authors (shorter visit time and more precise placement of pollen on insect body) and additionally the one of auto-deposition of pollen for selfing.

The experiments should be described in some more detail and using more precise wording. For example, there are places (e.g. line 257 and lines 295-299) where it is not clear if mobile vs. immobile stamen are compared on the side of the plant from which pollen is exported or on the side of the plant to which pollen is deposited (for example the latter is said in the legend to Figure 5, but from the text, the opposite seems to be the case). It is also not clear if self-pollen reception was tested in the absence or the presence of insects. Would stamens spontaneously snap in older flowers to ensure selfing in case they had not been visited?

The Discussion section is rather short, reflecting the relatively simple but interesting and novel results. Nevertheless, it seems there must be other literature about the effects of mobile stamens or at least the mechanism could be compared with other mechanisms assumed to reduce ineffective insect pollination in flowering plants or potentially enhance auto-deposition of pollen for selfing (relevant also because there was no difference in pollination success between hand-pollinating selfing and outcrossing treatments). Furthermore, the Discussion could point out where to go from the present "natural history" to more comprehensive studies in the model system. Finally, for this type of study, it would be useful to briefly discuss limitations.

Generally, the manuscript is well written and the analyses of the simple experiments are fine as far as it can be judged based on the brief descriptions.

Reviewer #2 (Recommendations for the authors):

This is an interesting report on the functional consequences and adaptive significance of a phenomenon scattered throughout the angiosperm clade: "explosive" snapping movement of stamens in response to being touched. Using four species of Berberidaceae, this study provides the first experimental evidence that rapid stamen movement and active pollen placement increase pollen dispersal to stigmas of other flowers. There is also evidence that the stamen-movement system results in pollen placement being more accurate with respect to sites of stigma contact and pollen being dispensed to a larger number of pollinators. These are all valuable insights into how natural selection has favored the evolution of complex floral behavior.

The writing is reasonably clear for the most part, although the sentence structure could be improved in many places. Analyses appear to be appropriate (although more information explaining and justifying the statistical approaches would be desirable). The results and conclusions appear to be valid.

Specific comments

1. Graphic-abstract caption and in main abstract: "Stamens only snap forward if their filament basis is touched by an insect tongue" implies that artificial stimulation (e.g. with a toothpick) doesn't trigger stamen movement. I don't think you mean this, and if not, rephrase.

2. Introduction line 5. "intended" must be in quotes or, better, not used at all.

3. Introduction, 1st paragraph. "Flowers are therefore under selection to 'pay' visitors as much as possible by nectar" is potentially misleading. Heinrich and Raven (Science 1972) argued that nectar production/standing crop would not be maximized, but rather optimized to promote pollinator movement.

4. Introduction, 2nd paragraph. I think you mean "base", not "basis".

5. Materials and methods a, paragraph 2. The preferred word is "pinned" not "needled."

6. Materials and methods c, 1st sentence. Don't use both passive voice ("were immersed") and active voice ("this blocked") in the same sentence. Change to, e.g., "all stamen movement was blocked…"

7. Results a, last line. "selfed" is ambiguous. I think you mean "manually self-pollinated".

8. Results f (and Statistical Methods section). I was unclear as to what assumptions and tests were used to assess the significance of differences in proportions of flowers receiving dyed pollen, e.g. "Of 400 nearby flowers in B. jamesiana, 75 received pollen from SM flowers and 40 from SI flowers, again indicating that mobile stamens donated pollen to more flowers (75/400 vs 40/400, G = 27.81, p < 0.001)." G-test doesn't explain the model or the assumptions, just implies log-likelihood. Is this assuming Normal or Poisson distributions or some nonparametric goodness of fit? The first doesn't seem appropriate and the latter yields a P-value slightly > 0.001 by my quick calculation. This needs a better explanation in the Method section. Because differential dispersal of pollen by motile vs. immobilized stamens is, arguably, the single most important result, its statistical validity should be made crystal clear.

9. The Discussion would benefit from editing and tighter organization. The language could be improved, removing awkward constructions. The discussion should perhaps start with a brief, well-organized summary of the major results (possibly enumerated), followed by comparisons with other species and studies.

Reviewer #3 (Recommendations for the authors):

Pollen, containing male gametes of flowering plants, functions in two roles, reproduction for plants and food resources for many insects, especially bees and hoverflies. From the plant aspect, the high fitness should achieve by dispersal pollen grains by the vector to the receptive stigmas of conspecific flowers; for the floral visitor aspect, the high benefit should collect more pollen grains by visiting fewer flowers. This is the pollen dilemma in pollination ecology. Many complex floral traits (concealed pollen grains, pollen/stamen mimicry) or floral behavior (floral part movement, pollen packaging/dispensing) could be driven by solving the "pollen dilemma". In this study, Li et al., presented a well-written paper with well-designed experiments to test the behavior and function of stamen movement in Berberis. The clever experiment design by using alcohol to immobilize stamens made the control and manipulating experiments possible. They found after immobilized stamens, bees visited flowers longer, but removed and deposited fewer pollen grains lowering plant fitness. Therefore, stamen movement drove insects move among flowers, and removed and deposited more pollen grains to conspecific flowers. In my opinion, this is an excellent example to illustrate how "floral trait/behavior" evolves to solve the "pollen dilemma".

The authors concluded that "These results demonstrate another mechanism by which plants simultaneously meter out their pollen and reduce pollen theft." I partly agree with this. The evidence to support pollen meter out is very strong, but the evidence to support "reduce pollen theft" is not strong enough.

The authors asked four questions, "(1) Are Berberis flowers with mobile stamens visited by the same types of insects and at the same rates as flowers with immobilized stamens? (2) Do flowers with mobile stamens have higher maternal and/or paternal success than those with immobilized stamens? (3) Do mobile stamens contribute to pollinator filtering by eliciting different behaviors in different insect taxa? And (4) is berberine, an alkaloid with antifeedant activity against herbivores that are found in Berberis leaves, also present in Berberis pollen or nectar?", in my opinion, questions (1) and (2) were well addressed in this research. Question (3) was not well addressed by the experiments. Question (4) was descriptive, there is a potential association with questions (1) and (2) but not well explained and tested.

Another weakness is the discussion selection. The authors did not fully discuss the novelty and application of key findings to floral trait/behavior evolution in a larger sense. The order of points of discussion also needs to be re-organized following questions and main results. For example, only one sentence talked about floral visitor filtering related to question (3).

Using alcohol to immobilize stamens is very important, should expand more to explain how alcohol immobilizes stamen movement in Introduction and Discussion.

The time of stamen movement should compare with the flower handling time. Is the time stamen movement always shorter than the time of flower handling by insects?

Another limitation of this study is that we still don't know if such floral behavior drive insects to move among individual plant. If such behavior only drives insects to move among flowers within the same plant, it cannot increase the diversity of paternal donors. Since a plant of Berberis can have many many flowers, it is not easy to test plant fitness at an individual plant level. Therefore, it is worth testing the function of stamen movement at individual plant level in the future using a model species producing countable numbers of flowers.

1. I found Figure 1B is very confusing. I checked Anthophora waltoni, see the following two references, I feel the bee in Figure 1B is so different with A. waltoni in the references. I suggest you should let your entomologist double-check the identification.

Mohamed Shebl, Li Qiang, Victor H. Gonzalez "Nesting Behavior, Seasonality, and Host Plants of Anthophora waltoni Cockerell (Hymenoptera: Apidae: Anthophorini) in Yunnan, China," Journal of the Kansas Entomological Society, 87(4), 345-349

Zhenghua Xie, Mohamed A. Shebl, Dongdong Pan et al., " Synergistically positive effects of brick walls and farmlands on Anthophora waltoni populations ", Agricultural and Forest Entomology 22(4), pg. 328, (2020); doi:10.1111/afe.12384

2. This statement on Meliosma tenuis is not accurate, in the case of Meliosma tenuis, very few workers of bumblebees visit flowers, one potential reason is that because workers could not collect the explosively released pollen.

3. "Flowers are therefore under selection to 'pay' visitors as much as possible by nectar". Don't drop into the trap that "nectar is for the goodness of floral visitor", nectar production is selected for the plant's own fitness, but such fitness is realized by floral visitation.

4. "Replenishable nectar". However, not all plant species replenish nectar, see the following reference. Luo, E.Y., Ogilvie, J.E. and Thomson, J.D. (2014). Stimulation of flower nectar replenishment by removal: A survey of eleven animal-pollinated plant species. J. Pollin. Ecol., 12, 52-62.

Reviewer #4 (Recommendations for the authors):

The study brings attention to a long-standing and assumed function of snapping stamens in Berberis spp. It contains four objectives to which the methodological approach is standard and most are broadly supported in the literature, even though the suitability of some designs could be argued as suboptimal proof of concept for generalization of certain pollination conclusions. This works aims to prove that visitation doesn't differ when stamens are able or not of snapping, but that the snapping mechanism improves the pollen uptake and transfer to pollinators and thus improves fruit formation, while the signature alkaloid compound of Berberis julianae is not a detriment to the interaction.

The results support well the null hypothesis of pollination filtering as well as the exploration of berberine compound per tissue is supported by the data, although runs short on its discussion. The visitation objective is not fully supported as it is not well described if the reported insects are all the visitors or not, neither if there was time compartamentalization of their visits. The maternal/paternal success objective can be argued as it is a self-pollinating plant with a large pollen to ovule ratio and requires very little pollen for fertilization. The lack of identity of the plant offspring raises a question about the effectivity or necessity of pollinators when offspring genotyping is missing and not much known about zygotic barriers or pollen compatibility.

Strengths

By adopting a 4 species array of plants and insects, it brings strength to a more general function of snapping stamens.

The manipulation experiments to immobilize stamens are a good approach to test the snapping function, with creative methodological variations and additional experiments to discard possible confounding effects.

Weaknesses

The selective in-depth testing of a pair of species makes it harder to generalize the pollen success at the multispecies level.

Although observed in the field (Table S2) provides no further inside observation of the pollinator interactions in nature, their time of activity, or any other insect visitation.

Lack of biological description of the receptivity, fertility, perception, day-cycle, and snapping capacity of the flowers over the 5 days that are assumed to be opened for all species.

No specification is given about some methods, such as the reattachment of manipulated flowers and their age.

Discussion is limited to plant aspects and overlooks insect aspects, such as basic avoidance behaviour (indirect evidence of berberine), possible optimal foraging theory, and learning behaviour (normally assumed in bees). The announced "scarcity of pollen" and self-pollination are lightly discussed and not necessarily based on robust premises. This last is a highly complex theme usually tackled with self-incompatible species due to the difficulty to assign which factor contributes more to forming offspring.

Arguable discussion about dispersal, measured just within the plant. No context on the foraging distances of A. cerana and A. walthoni, or the real impact of pollen compatibility and cross-pollination.

The study can provide a utilitarian reference for other similar questions after certain realization of how general or specific are some conclusions based on the methods and experimental design.

The title states mobile stamens, the use of snapping stamens will be a more accurate term as stated in the manuscript.

Intro

The number of pollen grains is not necessarily few when compared to the number of ovules.

How similar were the insect visitors at each location? And how was it established previously which were visitors and which real pollinators?

Methods

About the visitation and handling time, how are these criteria defined to consider a real visitation and the start and end of handling time?

Why was not tested the snapping in all Berberis species?

Was the evaluation of pollinator interaction in enclosed conditions observed and recorded visually or any camera aid was used?

The self-pollination experiments, have been tried with different genotypes of B. julianae, or how this was assessed? Since the 20m criteria in field condition are quite arbitrary. By not knowing the seed dispersal range or genotype diversity is hard to say if 20m is a reasonable distance.

How was the bee tongue manipulated to make snap the stamens in the experiments with SI/SM?

What is a fine day? Please specify the range of conditions.

How were the removed flowers in the field for SI treatment reattached?

How long flowers of Berberis are open in GH and field? Do reaction time for snapping vary on time?

How was the nectar estimated, by weight or volume, since a 10ul tip does not have so much graduation?

Why were selected the time windows for observation between 9-12 and 12-16, and so on? Is this related to the insect time window of activity?

How to distinguish with certainty selfing from cross-pollination?

In the field experiments with bees, how were other pollinators excluded? and how was recorded the interaction?

Citation missing at the sentence of leptokurtin distribution.

Why the pollen dispersal was evaluated only within the plant and in neighbouring plants?

Was a berberine standard used to confirm the HPLC readings?

Results

The use of "many fewer" can be shortened to just fewer.

What is the scale of vicinity on the flowers observed (f)?

I will suggest taking a look at papers on floral architecture and bee foraging behaviour. And also A. cerana mean flying distance for foraging.

Were other plants evaluated for stained pollen beyond the geitonogamy range?

The berberine response will require a proper electrophysiology test or choice assay or EAG.

Discussion

The proportion of pollen vs ovules, would not be considered scarce.

What about the tasting capacity and learning behaviour of bees with respect to berberine? It can be hypothesized that they will learn to avoid it.

It is mentioned in p.8 that selfed and cross-pollinated B. julianae don't differ, this argues against the pollen scarcity/limitation since very little pollen is needed to fertilize. Are all berberis so highly compatible or does genotype play a role in reducing self-compatibility?

Hard to say an increase in male plant reproductive success by the short distances evaluated.

Tables and figures

How are differences in visitation in Figure 3 A' explained?

Table S1 could profit with a PCA or PCOA.

Table S3 legend, to which species refers? seems a bad modification of the previous table legend.

eLife. 2022 Oct 11;11:e81449. doi: 10.7554/eLife.81449.sa2

Author response

Essential revisions:

1) The different experiments need to be described in more detail both with regard to the design, procedure, measurements, and analyses. Provide more biological description of the receptivity, fertility, perception, day-cycle, and snapping capacity of the flowers over the 5 days that they are assumed to be opened for all species, including potential auto-deposition by spontaneous snapping.

We have done that and also have clarified which species was observed for how long and where. The experiments are now described more fully and more clearly.

2) Of the four hypotheses the first two have been tested with good experimental designs and statistical power (pollen export from mobile vs. immobile stamens). However, this was not the case for the other two (pollinator filtering, presence or effect of berberine in pollen on pollinators). This should be acknowledged and discussed throughout the manuscript.

As we had written (page 9, lines 355-357, submitted version), “We also did not find a difference between flies and bees in their reaction to the snapping of stamens: Both visitor types left flowers more quickly after being hit by stamens, and stamen snapping therefore did not result in visitor filtering.” Our results thus do not support the hypothesis that mobile stamens contribute to pollinator filtering. We are now stating the three hypotheses tested more clearly in the Introduction and Discussion. Our Table S6 shows the berberine presence in Berberis pollen, but not nectar (cf. reply 43), however, any insects’ possible reaction to berberine is not one of the three hypotheses (as now stated more clearly), along with how our data address, support or reject them.

3) Check that the entomology is correct, including species identity and natural history. Also, be careful regarding the interpretation of pollen removal as indicative of berberine content in pollen.

We are very grateful to the reviewer for his comment on the bee shown in Figure 1B. We asked Dr. Michael Orr, an Anthophora expert, to check all our bee photos, and he identified the bee shown in Figure 1B as very likely Habropoda sichuanensis Wu, 1986. This identification was then confirmed (based on pinned specimens) by the first author and another Chinese colleague. Importantly, however, Anthophora waltoni is also among the pollinators, based on other pinned specimens and photos. We have corrected this throughout the text. We also consulted Dr. Antti Haarto, Zoological Museum, Section of Biodiversity and Environmental Science, Department of Biology, University of Turku, Finland, an expert on the syrphid genus Sphaerophoria, and he determined that the fly for which we had used that name is not a Sphaerophoria instead a species of Meliscaeva. Our other fly, Rhingia campestris, appears correctly identified.

Regarding the berberine in the pollen, please see Table S6 in our manuscript (cf. reply 43).

4) The Discussion should be improved by better synthesizing the different experiments into a general narrative and referring more extensively to general theory and other work on pollination aspects considered in this paper. Also, point out limitations and what would have to be done next to get a more comprehensive and mechanistic understanding of the function of mobile stamens (including pollinator behavior and niche separation as well as pollination results such as increased outcrossing rate or distance).

We now start the Discussion with a short summary about our main findings, and we now end it with a paragraph placing our findings in the context of other work on mobile stamens.

Reviewer #1 (Recommendations for the authors):

Besides the main results whether mobile stamens could "filter" visits according to pollinator species identity could not be confirmed and whether berberine affects pollinator behavior could only weakly be inferred from observation of pollen removal from bee tongues. It also remains to be shown whether mobile stamens additionally have a function in ensuring auto-deposition of pollen on stigmas for selfing and whether they increase seed production and offspring fitness of the plant.

While the experiments carried out are fine as far as they go, they are somewhat haphazardly planned using different species and places to test different partial questions relevant to the whole story. There is also some intrinsic confounding between different possible functions of mobile stamens, two mentioned by the authors (shorter visit time and more precise placement of pollen on insect body) and additionally the one of auto-deposition of pollen for selfing.

The experiments should be described in some more detail and using more precise wording. For example, there are places (e.g. line 257 and lines 295-299) where it is not clear if mobile vs. immobile stamen are compared on the side of the plant from which pollen is exported or on the side of the plant to which pollen is deposited (for example the latter is said in the legend to Figure 5, but from the text, the opposite seems to be the case). It is also not clear if self-pollen reception was tested in the absence or the presence of insects. Would stamens spontaneously snap in older flowers to ensure selfing in case they had not been visited?

Yes, selfing in Berberis is an interesting topic. As we had stated (page 4, lines 166-168, in the original version; now line 180)

“To examine intra-flower selfing induced by the stamen movement, we counted pollen grains deposited per stigma under a light microscope in 16 bagged flowers in which we had triggered one stamen by a needle.”

As shown in Figure S4, spontaneous autogamy does occur in this species, but its fruit/seed sets were both much lower (around 50%) compared to open or outcrossed pollination. We also state clearly (line 253)

“Self-pollen receipt by stigmas of B. julianae after a single stamen movement (14 ± 3 grains, N = 16) was only 6% of the pollen receipt resulting from a single visit by the most common visitors, Apis cerana, which deposited between 230 and 260 grains (section (d)) and roughly 1% of the pollen grains of a single anther with its two pollen sacs (ca. 1220 grains), indicating that intra-flower self-pollination mediated by the stamen movements plays a minor role in total pollen receipt.”

The Discussion section is rather short, reflecting the relatively simple but interesting and novel results. Nevertheless, it seems there must be other literature about the effects of mobile stamens or at least the mechanism could be compared with other mechanisms assumed to reduce ineffective insect pollination in flowering plants or potentially enhance auto-deposition of pollen for selfing (relevant also because there was no difference in pollination success between hand-pollinating selfing and outcrossing treatments). Furthermore, the Discussion could point out where to go from the present "natural history" to more comprehensive studies in the model system. Finally, for this type of study, it would be useful to briefly discuss limitations.

As explained above (reply 4), we now end it with a paragraph placing our findings in the context of other work on mobile stamens, some of it by professor Shuang-Quan Huang, for example, on Parnassia (Armbruster et al., 2014).

Generally, the manuscript is well written and the analyses of the simple experiments are fine as far as it can be judged based on the brief descriptions.

Reviewer #2 (Recommendations for the authors):

This is an interesting report on the functional consequences and adaptive significance of a phenomenon scattered throughout the angiosperm clade: "explosive" snapping movement of stamens in response to being touched. Using four species of Berberidaceae, this study provides the first experimental evidence that rapid stamen movement and active pollen placement increase pollen dispersal to stigmas of other flowers. There is also evidence that the stamen-movement system results in pollen placement being more accurate with respect to sites of stigma contact and pollen being dispensed to a larger number of pollinators. These are all valuable insights into how natural selection has favored the evolution of complex floral behavior.

The writing is reasonably clear for the most part, although the sentence structure could be improved in many places. Analyses appear to be appropriate (although more information explaining and justifying the statistical approaches would be desirable). The results and conclusions appear to be valid.

Specific comments

1. Graphic-abstract caption and in main abstract: "Stamens only snap forward if their filament basis is touched by an insect tongue" implies that artificial stimulation (e.g. with a toothpick) doesn't trigger stamen movement. I don't think you mean this, and if not, rephrase.

Rephrased by dropping “only”.

2. Introduction line 5. "intended" must be in quotes or, better, not used at all.

Replaced by “produced”.

3. Introduction, 1st paragraph. "Flowers are therefore under selection to 'pay' visitors as much as possible by nectar" is potentially misleading. Heinrich and Raven (Science 1972) argued that nectar production/standing crop would not be maximized, but rather optimized to promote pollinator movement.

That’s a misreading of our statement, which is here also quoted out of context. We meant that flowers should ‘pay’ pollinators by nectar instead of pollen (where possible), not that they should produce a lot of nectar.

4. Introduction, 2nd paragraph. I think you mean "base", not "basis".

Corrected, thank you.

5. Materials and methods a, paragraph 2. The preferred word is "pinned" not "needled."

Corrected, thank you.

6. Materials and methods c, 1st sentence. Don't use both passive voice ("were immersed") and active voice ("this blocked") in the same sentence. Change to, e.g., "all stamen movement was blocked…"

Corrected, thank you.

7. Results a, last line. "selfed" is ambiguous. I think you mean "manually self-pollinated".

Corrected, thank you.

8. Results f (and Statistical Methods section). I was unclear as to what assumptions and tests were used to assess the significance of differences in proportions of flowers receiving dyed pollen, e.g. "Of 400 nearby flowers in B. jamesiana, 75 received pollen from SM flowers and 40 from SI flowers, again indicating that mobile stamens donated pollen to more flowers (75/400 vs 40/400, G = 27.81, p < 0.001)." G-test doesn't explain the model or the assumptions, just implies log-likelihood. Is this assuming Normal or Poisson distributions or some nonparametric goodness of fit? The first doesn't seem appropriate and the latter yields a P-value slightly > 0.001 by my quick calculation. This needs a better explanation in the Method section. Because differential dispersal of pollen by motile vs. immobilized stamens is, arguably, the single most important result, its statistical validity should be made crystal clear.

We discussed our statistical approach with a colleague who is teaching biostatistics. He suggested that a G–test of independence (McDonald 2014) would be appropriate, and we now performed a G–test of independence in all comparisons of the effects of stamen movement on pollen flow. The results did not change from what we reported in the earlier version, but we now report the significance based on the G-test.

9. The Discussion would benefit from editing and tighter organization. The language could be improved, removing awkward constructions. The discussion should perhaps start with a brief, well-organized summary of the major results (possibly enumerated), followed by comparisons with other species and studies.

We now start the Discussion with a paragraph enumerating our main results.

Reviewer #3 (Recommendations for the authors):

Pollen, containing male gametes of flowering plants, functions in two roles, reproduction for plants and food resources for many insects, especially bees and hoverflies. From the plant aspect, the high fitness should achieve by dispersal pollen grains by the vector to the receptive stigmas of conspecific flowers; for the floral visitor aspect, the high benefit should collect more pollen grains by visiting fewer flowers. This is the pollen dilemma in pollination ecology. Many complex floral traits (concealed pollen grains, pollen/stamen mimicry) or floral behavior (floral part movement, pollen packaging/dispensing) could be driven by solving the "pollen dilemma". In this study, Li et al., presented a well-written paper with well-designed experiments to test the behavior and function of stamen movement in Berberis. The clever experiment design by using alcohol to immobilize stamens made the control and manipulating experiments possible. They found after immobilized stamens, bees visited flowers longer, but removed and deposited fewer pollen grains lowering plant fitness. Therefore, stamen movement drove insects move among flowers, and removed and deposited more pollen grains to conspecific flowers. In my opinion, this is an excellent example to illustrate how "floral trait/behavior" evolves to solve the "pollen dilemma".

The authors concluded that "These results demonstrate another mechanism by which plants simultaneously meter out their pollen and reduce pollen theft." I partly agree with this. The evidence to support pollen meter out is very strong, but the evidence to support "reduce pollen theft" is not strong enough.

The authors asked four questions, "(1) Are Berberis flowers with mobile stamens visited by the same types of insects and at the same rates as flowers with immobilized stamens? (2) Do flowers with mobile stamens have higher maternal and/or paternal success than those with immobilized stamens? (3) Do mobile stamens contribute to pollinator filtering by eliciting different behaviors in different insect taxa? And (4) is berberine, an alkaloid with antifeedant activity against herbivores that are found in Berberis leaves, also present in Berberis pollen or nectar?", in my opinion, questions (1) and (2) were well addressed in this research. Question (3) was not well addressed by the experiments. Question (4) was descriptive, there is a potential association with questions (1) and (2) but not well explained and tested.

Another weakness is the discussion selection. The authors did not fully discuss the novelty and application of key findings to floral trait/behavior evolution in a larger sense. The order of points of discussion also needs to be re-organized following questions and main results. For example, only one sentence talked about floral visitor filtering related to question (3).

Using alcohol to immobilize stamens is very important, should expand more to explain how alcohol immobilizes stamen movement in Introduction and Discussion.

The time of stamen movement should compare with the flower handling time. Is the time stamen movement always shorter than the time of flower handling by insects?

Another limitation of this study is that we still don't know if such floral behavior drive insects to move among individual plant. If such behavior only drives insects to move among flowers within the same plant, it cannot increase the diversity of paternal donors. Since a plant of Berberis can have many many flowers, it is not easy to test plant fitness at an individual plant level. Therefore, it is worth testing the function of stamen movement at individual plant level in the future using a model species producing countable numbers of flowers.

We are very grateful for these many good suggestions! Regarding the time of stamen movement, it is 0.44 ± 0.02 s in Berberis julianae, while the flower handling time by Apis cerana, the most common pollinator, is 4.919 ± 0.301 s and 5.017 ± 0.661 s by Habropoda sichuanensis (as reported in the manuscript). So, in this species, the stamen movement is much faster than the time that these bee species spent inside a flower. Regarding the genetic consequences of stamen mobility on male fitness, our pollen staining experiments provide empirical evidence for the greater donor distances achieved in flowers with functioning stamens compared to flowers with immobilized stamens. We don’t have genetic data and have therefore removed the word ‘fitness’ from ths manuscript and replaced it with ‘pollen flow distances’ and similar phrases (cf. reply 27)

1. I found Figure 1B is very confusing. I checked Anthophora waltoni, see the following two references, I feel the bee in Figure 1B is so different with A. waltoni in the references. I suggest you should let your entomologist double-check the identification.

Mohamed Shebl, Li Qiang, Victor H. Gonzalez "Nesting Behavior, Seasonality, and Host Plants of Anthophora waltoni Cockerell (Hymenoptera: Apidae: Anthophorini) in Yunnan, China," Journal of the Kansas Entomological Society, 87(4), 345-349

Zhenghua Xie, Mohamed A. Shebl, Dongdong Pan et al., " Synergistically positive effects of brick walls and farmlands on Anthophora waltoni populations ", Agricultural and Forest Entomology 22(4), pg. 328, (2020); doi:10.1111/afe.12384

Please see reply 2 above. You were correct that the bee on Figure 1B was not Anthophora waltoni.

2. This statement on Meliosma tenuis is not accurate, in the case of Meliosma tenuis, very few workers of bumblebees visit flowers, one potential reason is that because workers could not collect the explosively released pollen.

We corrected our statement (line 431) to ‘nectar-seeking bumblebee drones,’ based on Wong Sato and Kato’s conclusion (p. 537):

“The dominant flower visitors were nectar-seeking drones of the bumblebee species Bombus ardens (Apidae). The drone’s behavior, pollen attachment on their bodies, and fruit set of visit-restricted flowers suggest that they are the only agent triggering the explosive pollen release mechanism, and are the main pollinator of M. tenuis.”

3. "Flowers are therefore under selection to 'pay' visitors as much as possible by nectar". Don't drop into the trap that "nectar is for the goodness of floral visitor", nectar production is selected for the plant's own fitness, but such fitness is realized by floral visitation.

We did not fall into any trap about nectar production (this assumption by the reviewer is almost condescending). Please see reply 9.

4. "Replenishable nectar". However, not all plant species replenish nectar, see the following reference. Luo, E.Y., Ogilvie, J.E. and Thomson, J.D. (2014). Stimulation of flower nectar replenishment by removal: A survey of eleven animal-pollinated plant species. J. Pollin. Ecol., 12, 52-62.

We have now added “usually” before “replenishable.” The point here is that pollen is non-replenishable – ever. We agree with the reviewer’s general point, however, and one of us has even published on rewardless flowers: Renner, S. S. 2006. Rewardless flowers in the angiosperms and the role of insect cognition in their evolution. In N. M. Waser and J. Ollerton (eds.), Plant-Pollinator Interactions: From Specialization to Generalization, pp. 123-144. Univ. of Chicago Press, Chicago.

Reviewer #4 (Recommendations for the authors):

The study brings attention to a long-standing and assumed function of snapping stamens in Berberis spp. It contains four objectives to which the methodological approach is standard and most are broadly supported in the literature, even though the suitability of some designs could be argued as suboptimal proof of concept for generalization of certain pollination conclusions. This works aims to prove that visitation doesn't differ when stamens are able or not of snapping, but that the snapping mechanism improves the pollen uptake and transfer to pollinators and thus improves fruit formation, while the signature alkaloid compound of Berberis julianae is not a detriment to the interaction.

The results support well the null hypothesis of pollination filtering as well as the exploration of berberine compound per tissue is supported by the data, although runs short on its discussion. The visitation objective is not fully supported as it is not well described if the reported insects are all the visitors or not, neither if there was time compartamentalization of their visits. The maternal/paternal success objective can be argued as it is a self-pollinating plant with a large pollen to ovule ratio and requires very little pollen for fertilization. The lack of identity of the plant offspring raises a question about the effectivity or necessity of pollinators when offspring genotyping is missing and not much known about zygotic barriers or pollen compatibility.

Strengths

By adopting a 4 species array of plants and insects, it brings strength to a more general function of snapping stamens.

The manipulation experiments to immobilize stamens are a good approach to test the snapping function, with creative methodological variations and additional experiments to discard possible confounding effects.

Weaknesses

The selective in-depth testing of a pair of species makes it harder to generalize the pollen success at the multispecies level.

Although observed in the field (Table S2) provides no further inside observation of the pollinator interactions in nature, their time of activity, or any other insect visitation.

Lack of biological description of the receptivity, fertility, perception, day-cycle, and snapping capacity of the flowers over the 5 days that are assumed to be opened for all species.

No specification is given about some methods, such as the reattachment of manipulated flowers and their age.

Discussion is limited to plant aspects and overlooks insect aspects, such as basic avoidance behaviour (indirect evidence of berberine), possible optimal foraging theory, and learning behaviour (normally assumed in bees). The announced "scarcity of pollen" and self-pollination are lightly discussed and not necessarily based on robust premises. This last is a highly complex theme usually tackled with self-incompatible species due to the difficulty to assign which factor contributes more to forming offspring.

Arguable discussion about dispersal, measured just within the plant. No context on the foraging distances of A. cerana and A. walthoni, or the real impact of pollen compatibility and cross-pollination.

The study can provide a utilitarian reference for other similar questions after certain realization of how general or specific are some conclusions based on the methods and experimental design.

The title states mobile stamens, the use of snapping stamens will be a more accurate term as stated in the manuscript.

We changed the title to “Touch-sensitive stamens enhance pollen dispersal by scaring away visitors”

Intro

The number of pollen grains is not necessarily few when compared to the number of ovules.

Good point. We now provide the ratio of pollen grains to ovules in the studied species, viz. 2379 ± 49 in Berberis julianae and 3405 ± 78 in Berberis jamesiana.

How similar were the insect visitors at each location? And how was it established previously which were visitors and which real pollinators?

As stated, we studied

1) Berberis julianae C.K.Schneider in a field located at 29°52′26″N, 105°30′32″E, 427 m above sea level, in southeast of Anyue County, Sichuan Province, China.

2) B. jamesiana Forrest and W.W.Smith at Shangri-La Alpine Botanical Garden (N 27°54′05″, E 99°38′17″, 3300-3350 m above sea level), in Yunnan Province, Southwestern China.

3) B. forrestii Ahrendt at the Shangri-La field station,

4) Mahonia bealei (Fortune) Carrière in the Wuhan Botanical Garden (N 30°33′2″, E 114°25′48″, 23 m above sea level) in Hubei Province, China.

Given that each species was studied at a different location, we did not focus on comparing visitor spectra. There was no need to establish a priori which insects were visitors and which were pollen vectors because we have empirical observations on this point (especially given our experiments with stained pollen that was tracked among plants).

Methods

About the visitation and handling time, how are these criteria defined to consider a real visitation and the start and end of handling time?

Why was not tested the snapping in all Berberis species?

As stated on page 5, line 213-219, and shown in Table S1, we tested the stamen bending time in all three Berberis species as well as the Mahonia species.

Was the evaluation of pollinator interaction in enclosed conditions observed and recorded visually or any camera aid was used?

We produced videos of the interactions, three of which accompany our paper.

The self-pollination experiments, have been tried with different genotypes of B. julianae, or how this was assessed? Since the 20m criteria in field condition are quite arbitrary. By not knowing the seed dispersal range or genotype diversity is hard to say if 20m is a reasonable distance.

Yes, our experiments might involve genetic siblings. Assessing genotypes and sib mating in Berberis was beyond the scope of our study. Please compare reply 16.

How was the bee tongue manipulated to make snap the stamens in the experiments with SI/SM?

To quantify pollen loads placed on insect bodies and stigmas of next-visited conspecific flowers, we held captured and anaesthetized bees between forceps and made their tongues contact the filament bases in SI or SM flowers. This was described in the Supplementary Methods, but we now also described it in the main text’s M and M section.

What is a fine day? Please specify the range of conditions.

A sunny day (now stated, in response to reviewer #3).

How were the removed flowers in the field for SI treatment reattached?

As shown in Figure 1H, we inserted the pedicels of SI flowers into an inflorescence. We now state this clearly in the M and M section of the main text

How long flowers of Berberis are open in GH and field? Do reaction time for snapping vary on time?

Each flower lasts for about 4-5 days in Berberis julianae. We did not study changes in the stamen reaction ( = bending) time over several days, but do report Inward movement time (s), Interval time (s), and Outward movement time (s) in Table S1.

How was the nectar estimated, by weight or volume, since a 10ul tip does not have so much graduation?

The nectar volume per flower of B. julianae was estimated by using clean 10 µL microliter syringes (Agilent Technologies Inc, USA) to extract nectar drops from bagged flowers. This was and is described in the Suppl. Mats.

Why were selected the time windows for observation between 9-12 and 12-16, and so on? Is this related to the insect time window of activity?

Yes, the observation times reflect the insects’ main activity times.

How to distinguish with certainty selfing from cross-pollination?

Selfing refers to receipt of a flower’s own pollen grains. Cross-pollination refers to receipt of pollen grains from a different Berberis shrub. As explained above, data on genetic structure (and possible sib mating) were beyond the scope of this study.

In the field experiments with bees, how were other pollinators excluded? and how was recorded the interaction?

In the field experiments with bees, we did not exclude other pollinators, since there were too few non-bee visits to visit the treated flowers (which in one experiment were harvested after single bee visits; as described in M and M).

Citation missing at the sentence of leptokurtin distribution.

We now added a review to support our statement “Previous studies have indicated that pollen flow mediated by generalist insects usually occurs within meters with a highly leptokurtic distribution.” Williams, 2001, which concluded “Studies of pollen and gene flow within patches/crops [..-], whether wind- or insect-pollinated, generally describe highly leptokurtic distributions of pollen from the source plants, with levels decreasing rapidly within a few metres of the source to a low level at which they remain for a much longer distance.

Why the pollen dispersal was evaluated only within the plant and in neighbouring plants?

Because detecting the stained pollen grains in the vicinity of the donor plant was difficult. We checked hundreds of stigmas at the distances mentioned in the manuscript and Tables S3 and S4.

Was a berberine standard used to confirm the HPLC readings?

Yes, a berberine standard was used to confirm the HPLC readings.

Results

The use of "many fewer" can be shortened to just fewer.

Done.

What is the scale of vicinity on the flowers observed (f)?

About 25 to 100 cm from the source as indicated in tables S3 and S4. We have now added this information in the sentence where the word ‘vicinity’ first appears.

I will suggest taking a look at papers on floral architecture and bee foraging behaviour. And also A. cerana mean flying distance for foraging.

Thank you for this suggestion.

Were other plants evaluated for stained pollen beyond the geitonogamy range?

We checked hundreds of stigmas (specifically up to 994 and 733) on different shrubs at the distances mentioned in the manuscript and Tables S3 and S4.

The berberine response will require a proper electrophysiology test or choice assay or EAG.

Why? We observed bees cleaning Berberis pollen from their bodies, and this is all that we report, along with the berberine concentrations in leaves, flowers, pollen, and nectar (Table S6).

Discussion

The proportion of pollen vs ovules, would not be considered scarce.

We revised as “Especially in flowers with open-access pollen grains”.

What about the tasting capacity and learning behaviour of bees with respect to berberine? It can be hypothesized that they will learn to avoid it.

We agree, but have no data on these points.

It is mentioned in p.8 that selfed and cross-pollinated B. julianae don't differ, this argues against the pollen scarcity/limitation since very little pollen is needed to fertilize. Are all berberis so highly compatible or does genotype play a role in reducing self-compatibility?

These interesting questions are outside the scope of our study.

Hard to say an increase in male plant reproductive success by the short distances evaluated.

We beg to differ. In flowers with immobilized stamens, the commonest bee species stayed up to 3.6x longer, yet removed 1.3x fewer pollen grains and deposited 2.1x fewer grains on stigmas per visit. Mobile stamens exported their pollen to significantly more flowers.

Tables and figures

How are differences in visitation in Figure 3 A' explained?

Figure 3 A' shows the visitation rates of Apis cerana to SM and SI flowers in the field. As described in M and M, SI flowers were cut off and their pedicels inserted into little vials filled with alcohol, and after a certain time, this probably reduced their nectar production.

Table S1 could profit with a PCA or PCOA.

Table S1 floral traits (mean ± SE) and duration of stamen movements in Berberis jamesiana, B. julianae, B. forrestii, and Mahonia bealei. Different superscript letters indicate significant differences; N = number of sampled flowers; － not measured. We do not understand why a principal component analysis is needed here.

Table S3 legend, to which species refers? seems a bad modification of the previous table legend.

We have dropped Table S3.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file 1. Floral traits (mean ± SE) and duration of stamen movements in Berberis jamesiana, B. julianae, B. forrestii, and Mahonia bealei.

elife-81449-supp1.docx^{(15.7KB, docx)}

Supplementary file 2. Foraging behaviors of two types of major insect visitors to Berberis julianae.

elife-81449-supp2.docx^{(16.4KB, docx)}

elife-81449-supp3.docx^{(13.2KB, docx)}

elife-81449-supp4.docx^{(12.8KB, docx)}

Supplementary file 5. Concentrations of berberine (mean ± SE) in different tissues of Berberis julianae under generalized linear models.

elife-81449-supp5.docx^{(13KB, docx)}

MDAR checklist

elife-81449-mdarchecklist1.docx^{(99.2KB, docx)}

Data Availability Statement

All data are shown in the main text and the tables at the end of the main text.

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PERMALINK

Touch-sensitive stamens enhance pollen dispersal by scaring away visitors

Deng-Fei Li

Wen-Long Han

Susanne S Renner

Shuang-Quan Huang

Roles

Abstract

Introduction

Figure 1. Flower traits, foraging behavior of visitors, and manipulations of stamen movements in Berberis julianae, which has stamens characterized by a touch-sensitive rapid movement toward the flower center.

Figure 1—figure supplement 1. Flower manipulations and experimental floral arrays in Berberis julianae.

Figure 1—figure supplement 2. Habit, floral traits, developing berries, and feeding behavior of flower visitors in three Berberidaceae species whose stamens are touch-sensitive.

Results

Touch-sensitive stamen movements and experimental selfing and outcrossing

Figure 2. Fruit and seed set (mean ± Standard Error) after four pollination treatments in Berberis julianae.

Flower visitors and pollinators

Tests for a possible confounding effect of the alcohol treatment on visitor behavior

Figure 3. Visitation rates (A, C) and handling times (B, D) of Apis cerana in four treatments of flowers of Berberis julianae.

Effects of forward-snapping stamens on visitor behavior and pollination in the field and under enclosed conditions

Figure 4. Comparisons of six parameters (mean ± Standard Error) in Berberis julianae to examine the effects of stamen movements on insect visitor foraging behavior and their roles in pollination.

Effects of mobile stamens on pollen receipt in experimental visits

Figure 5. Effects of stamen forward-snapping on pollen placement on the pollinator body and pollen deposition on stigmas after single visits by Apis cerana (A, C) and the anthophorid bees (B, D).

Effects of mobile stamens on pollen export and receipt in the field

Figure 6. Number of stained pollen grains deposited on the stigmas from control flowers with mobile stamens (open circles) and from flowers with experimentally immobilized stamens (closed circles) in Berberis julianae.

Figure 6—figure supplement 1. Number of stained pollen grains deposited on the stigmas from control flowers with mobile stamen (open circles) and from flowers with experimentally immobilized stamens (closed circles) in Berberis jamesiana.

Berberine content in B. julianae leaves, petals, pollen, and nectar

Discussion

Conclusion

Materials and methods

Plant and insect study species

Alcohol as an inhibitor of the stamen movement, and tests for confounding the effects of the alcohol treatment on visitor behavior

Video 1. Stamens of Berberis julianae become touch-insensitive after the flower pedicels had been immersed in 75% alcohol for 35 min.

Video 2. Stamens of Mahonia bealei become touch-insensitive after the flower pedicels had been immersed in 75% alcohol for 30 min.

Fruit and seed set after self-pollination vs. cross-pollination, and contribution of stamen forward-snapping to selfing

Effect of stamen bending on visitor behavior and pollen export and import

Pollen-tracking experiments to quantify the effects of stamen forward-snapping on pollen export distances

Statistical analyses

Acknowledgements

Appendix 1

Measurements of floral traits in Berberis and Mahonia species

Testing protocols for best alcohol treatments to block the stamen movement

Floral visitors and effect of stamen bending on their behavior and pollen export and import

Pollen export/import after single visits

Pollen-tracking experiments to quantify the effects of stamen forward-snapping on pollen export distances

Measurement of chemical defense in pollen and nectar

Funding Statement

Contributor Information

Funding Information

Additional information

Competing interests

Author contributions

Additional files

Data availability

References

Editor's evaluation

Bernhard Schmid

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Decision letter

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Author response

Associated Data

Supplementary Materials

Data Availability Statement

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