Do Silene species with exposed stigmas tolerate interference by heterospecific pollen?

PREMISE: Co-flowering species that have not evolved an avoidance mechanism may have tolerance to heterospecific pollen (HP) deposition as an adaptive strategy to minimize any deleterious effects of HP transfer, but empirical evidence for the tolerance hypothesis remains scarce. METHODS: To estimate the potential effects of heterospecific pollen deposition (HPD) on female reproductive success, we counted conspecific (CP) and HP pollen grains deposited on stigmas and assessed subsequent seed set of both open- and hand-pollinated flowers in three co-flowering Silene species with exposed stigmas that usually received numerous HP grains on the elongated receptive area. RESULTS: The percentage of HP grains per flower (HP%) varied from 16.6% to 43.0% among three species. Silene chungtienensis had lower HP%, and the CP-HP relationship was neutral; S. gracilicaulis and S. yunnanensis had a relatively higher HP% with a positive CP-HP relationship. The effects of CP and HP number on natural seed set were positive for all three species, but HP% had stronger negative effects in S. chungtienensis and S. gracilicaulis. In hand-pollinated flowers of the three Silene species, seed set did not decrease with HP whether CP was in excess or insufficient, indicating no negative effects of HPD on seed production. CONCLUSIONS: Consistent with the tolerance hypothesis, our results indicated that species with higher HP interference are likely to be tolerant to an increase in HP%. These species with generalist-pollinated flowers and exposed large stigmas may benefit from an increase of conspecific pollen deposition, despite the associated increase in heterospecific pollen deposition.

Pollen-feeding behavior of diverse insects on Geranium delavayi, a flower with large, accessible pollen grains.

PREMISE: Why have pollen grains evolved to be exceptionally large in some species? Pollen-feeding hypothesis suggests that if the proportion of pollen amounts for feeding is reduced in a flower, the low allocation to pollen number would allow pollen grains to be larger. METHODS: To examine whether species with large pollen grains experience low pollen consumption, the behavior of insects feeding on nectar and pollen was observed and pollen transfer efficiency was estimated for four visitor types in Geranium delavayi. To see whether bees actively collected pollen, the numbers of grains in pollen baskets and on the body were compared. Both nutritional value (total protein and lipid) and chemical defense (phenolic metabolites) in pollen against pollen feeders were measured. RESULTS: Bumblebees and honeybees foraged for nectar, rarely groomed pollen into corbiculae, and had >5× higher pollen transfer efficiency than smaller solitary bees and flies, which were pollen eaters that removed more pollen but deposited less. Pollen grains were characterized by low protein and high lipid content with a low protein-lipid ratio, an unfavorable combination for bumblebees. Three secondary metabolites were significantly higher in pollen grains (7.77 mg/g) than in petals (1.08 mg/g) or in nectar (0.44 mg/g), suggesting stronger chemical defense in pollen. CONCLUSIONS: Our results indicated that large bees took nectar but little of the nutritionally poor and highly toxic pollen. These data support one prediction of the pollen-feeding hypothesis, that species with few and large pollen grains would also have low pollen-consumption rates.

Touch-sensitive stamens enhance pollen dispersal by scaring away visitors.

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.

Foraging behavior and pollination efficiency of generalist insects in an understory dioecious shrub Helwingia japonica.

PREMISE: It has been hypothesized that pollination success in animal-pollinated dioecious plants relies on opportunistic pollinators with no discrimination against female flowers. However, empirical studies of pollinator foraging behavior and pollination effectiveness in dioecious species are few. METHODS: To investigate potential pollinators in Helwingia japonica, a dioecious shrub with small, inconspicuous flowers, we compared floral visitors and visit frequency to female and male plants for three flowering seasons in two field populations in subtropical forests in southwest China. Pollen placement on the insect bodies of four groups (solitary bees and other bees, fungus gnats, and other flies) was compared, and insect foraging behavior was observed. Pollen removal and conspecific and heterospecific pollen deposition per visit were measured to compare pollination effectiveness among the four groups. RESULTS: Floral visitors usually did not discriminate between male and female flowers and did not gather pollen into collections. Our measurements of pollen transfer efficiency showed that solitary bees were the most effective pollinators with the highest conspecific pollen deposition. These insects seemed to be opportunistic visitors because pollen grains of H. japonica were distributed evenly over different regions of the visitor's body, and heterospecific pollen accounted for over 50% of total pollen loads on stigmas in the two populations. CONCLUSIONS: Our investigations indicated that potential pollinators were generalists and did not discriminate against female flowers, as predicted for dioecious species pollinated by insects. A perspective of pollen removal by floral visitors offers insights into the evolution of plant sexual systems.

Pollen grain size associated with pollinator feeding strategy.

Angiosperm pollen grain diameter varies greatly from a few microns to over 100, but the selective forces driving the interspecific variation in pollen size remain unclear. Although both pre- and post-pollination hypotheses have been proposed, empirical evidence remains scarce. Here we propose that visits by pollen-foraging pollinators have selected against large pollen grains. An association between pollinator behaviour and pollen grain size was confirmed by field studies of 80 flowering species in natural communities, showing that pollinators positively collected pollen in those species with relatively smaller pollen grains but rarely did so in species with larger ones. Allowing for the confounding effects of pollinator type, flower size or style length and pollen grain number, we found a significant effect of pollen-foraging behaviour on variation in pollen grain size, particularly in bee-pollinated plants. While these results suggest that many plant species whose pollen is collected or consumed by pollinators produce small pollen grains, it remains unclear whether pollen grain size is directly affected by pollinator foraging habit or indirectly mediated by pollen number trade-offs.

Pollinator effectiveness and importance between female and male mining bee (Andrena).

Bees are often considered to be effective pollinators in both agricultural and natural ecosystems but could be ineffective pollinators in that they collect large quantities of pollen for food provision but deliver little to stigmas. Male bees do not collect pollen to feed larvae, and their pollination role has been underappreciated. Here we compare pollination effectiveness, visit frequency and pollen foraging behaviour between female and male individuals of a mining bee, Andrena emeishanica, visiting a nectariferous spring flower (Epimedium pubescens). Female bees were observed to forage for both pollen and nectar, but male bees foraged only for nectar. Female bees had large hairy hind tibiae with conspicuous scopae, and nearly 90% of the pollen grains they collected went onto the hind legs. Male bees removed less pollen from anthers than female bees but deposited more pollen on stigmas per visit. The higher pollen transfer efficiency of male bees was due to 48.4% of pollen grains remaining ungroomed on the thorax and abdomen, available for stigma contact, but their visitation rate to flowers was much lower. Our results indicate that male solitary bees could transfer more pollen on the stigma per visit but were less important (transferred less pollen in total, because they made fewer visits per unit time) than females.

Color-matching between pollen and corolla: hiding pollen via visual crypsis?

Visual signals attractive to friends may also attract enemies.  The bright colors of anthers and pollen have generally been thought to attract pollinators. We hypothesize that visual crypsis of anthers, and particularly pollen, should be favored in flowering plants because protection from pollen collectors reduces the loss of male gametes. To understand adaptive strategies relating to the color of pollen, we measured the color of pollen, undehisced anther sacs, and their background, the corolla, with a spectrometer for 104 insect-pollinated flowering species from a natural community in Hengduan Mountains, southwest China. The colors of anthers, pollen and corollas were diverse in these species. The color diversity of exposed pollen was significantly higher than that of concealed pollen (i.e. where anthers are enclosed or shielded by corollas). The color contrast between pollen and corolla was significantly smaller in species with exposed pollen than in those with concealed pollen. Unlike anther color, exposed pollen color tended to match its background corolla color. Our phylogenetic comparative analysis showed contrasting effects of pollen color patterns between flowers with exposed pollen and those with concealed pollen, revealing a strategy of hiding pollen from pollen thieves via visual crypsis.

Bumblebee Rejection of Toxic Pollen Facilitates Pollen Transfer.

Many bees are effective pollen collectors; however, pollen grains collected by bees for larval food are lost for plant sexual reproduction. Recognition of these conflicting interests between bees and flowers is essential for understanding of reproduction for both bees and flowers [1-3]. Plant defense compounds in pollen may function to reduce pollen waste by deterring ineffective pollinators [4-6], but this hypothesis remains unexamined. Here, we provide evidence that secondary metabolites in pollen function as chemical defense by deterring some bees from gathering pollen. In two Dipsacus species, a defense compound, dipsacus saponin [7], occurs in pollen but not in nectar. We observed that bumblebees disliked grooming bitter-tasting pollen with a high saponin content. Manipulation of saponin concentrations in nectar and measurements of corbicular pollen showed that the bumblebee species differed in their tolerance to saponin. Those species susceptible to saponin groomed little Dipsacus pollen into their pollen loads, and their ungroomed pollen was observed to be effectively delivered to stigmas. By rewarding bees with edible nectar, but not pollen, plants solve the conflict of pollen partitioning between sexual and reward functions. Ungroomed toxic pollen on the bee body promotes pollen transfer efficiency, facilitating pollination.

Pollen aggregation by viscin threads in Rhododendron varies with pollinator.

Pollen grains can be dispersed singly or variously aggregated in groups. Whether the evolution of pollen aggregation is driven by the pollinator remains unexplored. We hypothesize that an extensive pollen aggregation is favored under a scarcity of pollinators. Variation in pollen aggregation by viscin threads in 13 Rhododendron species was measured as it is related to pollen removal in a visit. Visitation rates of functional pollinator groups that vary in their grooming behavior were investigated in each species. Pollen deposited on stigmas in the field was also sampled. Seven Rhododendron species were infrequently pollinated by low-intensity grooming animals, including birds, butterflies and moths. The other six species were more frequently pollinated by bees with a high intensity of pollen grooming. Bird- and Lepidoptera-pollinated species produced longer pollen-connecting threads that connected more pollen grains. Phylogenetically independent contrast analysis of the 13 species showed that pollinator visitation frequency was negatively related to amounts of pollen removal per visit but not to stigmatic pollen loads. The finding of interspecific patterns in pollen removal related to pollinator visitation frequency suggests pollinator-mediated selection on pollen packaging strategies, supporting the hypothesis of floral evolution via pollen export.

Heterostyly promotes compatible pollination in buckwheats: Comparisons of intraflower, intraplant, and interplant pollen flow in distylous and homostylous Fagopyrum.

PREMISE OF THE STUDY: Heterostyly, the reciprocal positioning of stigmas and anthers in different floral morphs, has long been thought to promote intermorph pollination. However, extensive intramorph pollination occurs commonly in heterostylous species, leading to recurrent questions about the functional and evolutionary significance of heterostyly. METHODS: To identify the sources of stigmatic pollen (autogamous [intraflower], geitonogamous [intraplant], vs. interplant), we emasculated either one flower or entire plants in experimental populations of the two closely related buckwheat species, distylous Fagopyrum esculentum and homostylous F. tataricum. Differences in pollen size allowed unambiguous identification of pollen on stigmas. RESULTS: Only 2.4% of F. tataricum pollen and 1.5% of F. esculentum pollen arrived successfully on compatible stigmas of other plants. In the former (homostylous) species, 71.3% of the pollen load on stigmas was autogamous, 10.8% was geitonogamous, and 17.9% was interplant. In the latter (distylous) species, 37.45% of the pollen on stigmas was autogamous, 13.8% was geitonogamous, 17.0% was intramorph, and 31.75% was intermorph. The amount of incompatible pollen arriving on stigmas was greatly decreased by both one-flower and whole-plant emasculations, and thus, the proportion of compatible pollen deposited increased with one-flower emasculation and increased even more with whole-plant emasculation. CONCLUSIONS: Our quantification of pollen-donor sources in these two species indicated that heterostyly in Fagopyrum esculentum provided a nearly 2-fold fitness advantage (in terms of compatible pollination) over expected (random) pollen transfers between morphs. Because of reduced herkogamy, the homostylous F. tataricum was highly autogamous.

Safe sites of pollen placement: a conflict of interest between plants and bees?

Plant stigmas and bee pollinators are competitors for pollen. Pollen placed on a pollinator's body can be picked up by conspecific stigmas or it can be collected by the pollinator as food. Hypothetically, one solution is for pollen to be placed on 'safe sites' on the pollinator's body, sites where the pollinator cannot easily remove it, leaving the pollen for stigmas. We compared 14 sites on the bumblebee body in terms of the ability of the bee to groom off fluorescent power, a dust that may be thought of as analogous to pollen. The safest sites were along the midline of the dorsal thorax, the dorsal abdomen, and the ventral abdomen. Next, we counted how much pollen is borne on the 14 sites by bees visiting one nectariferous and three nectarless Pedicularis species. In the four species, only 7, 26, 28, and 30% of pollen found on the bees were on safe sites. Finally, we observed that the 14 sites were contacted by stigmas of the four Pedicularis species; none of the most contacted sites were safe sites. Across all four Pedicularis species, pollen is mainly positioned on sites of the bee body that were beneficial for both the plant and the bee, not on sites detrimental to either of them. Our analysis showed that the conflict of interest between flowers and bees can be solved by cooperation. Pedicularis pollen is placed where it strengthens the mutualism between plants and pollinators.

A paradoxical mismatch between interspecific pollinator moves and heterospecific pollen receipt in a natural community.

Pollinators visiting multiple plant species may cause heterospecific pollen transfer (HPT). To test a null model that more pollinator interspecific moves result in higher HPT among interacting species, we quantified the comparative magnitudes of the two networks involving 14 co-flowering species in an alpine meadow in the eastern Himalaya, southwest China. Interspecific moves accounted for 4% of the total visits,whereas heterospecific pollen constituted 22% of the total stigmatic pollen loads. On average, plant species received interspecific moves and HPT from 6.9 and 9.7 other species, respectively. Although the two networks were largely concordant, 21.6% of interspecific move links were not correspondingly linked by HPT, and 44.1% of heterospecific pollen transfer links were not linked by moves. Plant species with more outgoing pollinator moves tended to disperse more of their own pollen to others, as expected. Surprisingly, our data reveal that plant species which received more pollinator moves from other species tended to receive less HP, implying that only species with low acceptance of HP were likely to permit frequent pollinator moves. These new findings unveil a paradoxical relation between pollinator interspecific moves and HP receipt, suggesting an adaptive strategy of co-flowering species that reduces deleterious effects of HPT.

Geographic consistency and variation in conflicting selection generated by pollinators and seed predators.

BACKGROUNDS AND AIMS: Floral traits that attract pollinators may also attract seed predators, which, in turn, may generate conflicting natural selection on such traits. Although such selection trade-offs are expected to vary geographically, few studies have investigated selection mediated by pollinators and seed predators across a geographic mosaic of environments and floral variation. METHODS: Floral traits were investigated in 14 populations of the bumble-bee-pollinated herb, Pedicularis rex, in which tubular flowers are subtended by cupular bracts holding rain water. To study potentially conflicting selection on floral traits generated by pollinators and florivores, stigmatic pollen loads, initial seed set, pre-dispersal seed predation and final viable seed production were measured in 12-14 populations in the field. KEY RESULTS: Generalized Linear Model (GLM) analyses indicated that the pollen load on stigmas was positively related to the exsertion of the corolla beyond the cupular bracts and size of the lower corolla lip, but so too was the rate of seed predation, creating conflicting selection on both floral traits. A geographic mosaic of selection mediated by seed predators, but not pollinators, was indicated by significant variation in levels of seed predation and the inclusion of two-, three- and four-way interaction terms between population and seed predation in the best model [lowest corrected Akaike Information Criterion (AICc)] explaining final seed production. CONCLUSIONS: These results indicate opposing selection in operation: pollinators generated selection for greater floral exsertion beyond the bracts, but seed predators generated selection for reduced exsertion above the protective pools of water, although the strength of the latter varied across populations.

Pre- and post-pollination interaction between six co-flowering Pedicularis species via heterospecific pollen transfer.

It remains unclear how related co-flowering species with shared pollinators minimize reproductive interference, given that the degree of interspecific pollen flow and its consequences are little known in natural communities. Differences in pollen size in six Pedicularis species with different style lengths permit us to measure heterospecific pollen transfer (HPT) between species pairs in sympatry. The role of pollen-pistil interactions in mitigating the effects of HPT was examined. Field observations over 2 yr showed that bumblebee pollinators visiting one species rarely moved to another. Heterospecific pollen (HP) comprised < 10% of total stigmatic pollen loads for each species over 2 yr, and was not related to conspecific pollen deposition. Species with longer styles generally received more HP per stigma. The pollen tube study showed that pollen from short-styled species could not grow the full length of the style of long-styled species. Pollen from long-styled species could grow through the short style of P. densispica, but P. densispica rarely received HP in nature. Flower constancy is a key pre-pollination barrier to HPT between co-flowering Pedicularis species. Post-pollination pollen-pistil interactions may further mitigate the effects of HPT because HP transferred to long styles could generally be effectively filtered.

Pollen size strongly correlates with stigma depth among Pedicularis species.

Darwin proposed that pollen size should be positively correlated with stigma depth rather than style length among species given that pollen tubes first enter the stigma autotrophically, then grow through the style heterotrophically. However, studies often show a positive relationship between pollen size and style length. Five floral traits were observed to be correlated among 42 bumblebee-pollinated Pedicularis species (Orobanchaceae) in which stigmas are distinct from styles. The phylogenetic independent contrast analysis revealed that pollen grain volume was more strongly correlated with stigma depth than with style length, consistent with Darwin's functional hypothesis between pollen size and stigma depth.

Buzz pollination in eight bumblebee-pollinated Pedicularis species: does it involve vibration-induced triboelectric charging of pollen grains?

BACKGROUND AND AIMS: Buzz pollination involves explosive pollen release in response to vibration, usually by bees. The mechanism of pollen release is poorly understood, and it is not clear which component of vibration (acceleration, frequency, displacement or velocity) is critical; the role of buzz frequency has been particularly controversial. This study proposes a novel hypothesis that explosive pollen release results from vibration-induced triboelectric charging. If it does, pollen release is expected to depend on achievement of a critical threshold velocity. METHODS: Eight sympatric buzz-pollinated species of Pedicularis that share bumblebee pollinator species were studied, giving a rare opportunity to compare sonication behaviour of a shared pollinator on different plant species. KEY RESULTS: Reconsidering previous experimental studies, it is argued that they establish the critical role of the velocity component of vibration in pollen release, and that when displacement is constrained by body size bees can achieve the critical velocity by adjusting frequency. It was shown that workers of Bombus friseanus assorted themselves among Pedicularis species by body size, and that bees adjusted their buzz/wingbeat frequency ratio, which is taken as an index of the velocity component, to a value that corresponds with the galea length and pollen grain volume of each species of Pedicularis. CONCLUSIONS: Sonication behaviour of B. friseanus differs among Pedicularis species, not only because worker bees assort themselves among plant species by body size, but also because bees of a given size adjust the buzz frequency to achieve a vibration velocity corresponding to the floral traits of each plant species. These findings, and the floral traits that characterize these and other buzz-pollinated species, are compatible with the hypothesis of vibration-induced triboelectric charging of pollen grains.

In the right place at the right time: Parnassia resolves the herkogamy dilemma by accurate repositioning of stamens and stigmas.

BACKGROUND AND AIMS: Spatial (herkogamy) and temporal (dichogamy) separation of pollen presentation and stigma receptivity have been interpreted as reducing interference between male and female functions in hermaphroditic flowers. However, spatial separation leads to a potential conflict: reduced pollination accuracy, where pollen may be placed in a location on the pollinator different from the point of stigma contact. METHODS: To understand better how herkogamous flowers resolve this conflict, a study was made of a subalpine herb, Parnassia epunctulata, the nectariferous flowers of which exhibit sequential anther dehiscence (staggered pollen presentation) and stamen movements; usually one newly dehisced anther is positioned each day over the central gynoecium, while the older stamens bend away from the central position. KEY RESULTS: The open flowers were visited by a variety of pollinators, most of which were flies. Seed set was pollinator-dependent (bagged flowers set almost no seeds) and pollen-limited (manual pollination increased seed set over open pollination). Analyses of adaptive accuracy showed that coordinated stamen movements and style elongation (movement herkogamy) dramatically increased pollination accuracy. Specifically, dehiscing anthers and receptive stigmas were positioned accurately in the vertical and horizontal planes in relation to the opposite sexual structure and pollinator position. By contrast, the spatial correspondence between anthers and stigma was dramatically lower before the anthers dehisced and after stamens bent outwards, as well as before and after the period of stigmatic receptivity. CONCLUSIONS: It is shown for the first time that a combination of movement herkogamy and dichogamy can maintain high pollination accuracy in flowers with generalized pollination. Staggered pollen and stigma presentation with spatial correspondence can both reduce sexual interference and improve pollination accuracy.

Pistillate flowers experience more pollen limitation and less geitonogamy than perfect flowers in a gynomonoecious herb.

Gynomonoecy, a sexual system in which plants have both pistillate (female) flowers and perfect (hermaphroditic) flowers, occurs in at least 15 families, but the differential reproductive strategies of the two flower morphs within one individual remain unclear. Racemes of Eremurus anisopterus (Xanthorrhoeaceae) have basal pistillate and distal perfect flowers. To compare sex allocation and reproductive success between the two flower morphs, we measured floral traits, pollinator preferences, and pollen movement in the field. Pollen limitation was more severe in pistillate flowers; bee pollinators preferred to visit perfect flowers, which were also capable of partial self-fertilization. Pollen-staining experiments indicated that perfect flowers received a higher proportion of intra-plant pollen (geitonogamy) than pistillate flowers. Plants with greater numbers of pistillate flowers received more outcross pollen. The differential reproductive success conformed with differential floral sex allocation, in which pistillate flowers produce fewer but larger ovules, resulting in outcrossed seeds. Our flower manipulations in these nectarless gynomonoecious plants demonstrated that perfect flowers promote seed quantity in that they are more attractive to pollinators, while pistillate flowers compensate for the loss of male function through better seed quality. These results are consistent with the outcrossing-benefit hypothesis for gynomonoecy.

Do specialized flowers promote reproductive isolation? Realized pollination accuracy of three sympatric Pedicularis species.

BACKGROUND AND AIMS: Interest in pollinator-mediated evolutionary divergence of flower phenotype and speciation in plants has been at the core of plant evolutionary studies since Darwin. Specialized pollination is predicted to lead to reproductive isolation and promote speciation among sympatric species by promoting partitioning of (1) the species of pollinators used, (2) when pollinators are used, or (3) the sites of pollen placement. Here this last mechanism is investigated by observing the pollination accuracy of sympatric Pedicularis species (Orobanchacae). METHODS: Pollinator behaviour was observed on three species of Pedicularis (P. densispica, P. tricolor and P. dichotoma) in the Hengduan Mountains, south-west China. Using fluorescent powder and dyed pollen, the accuracy was assessed of stigma contact with, and pollen deposition on, pollinating bumble-bees, respectively. KEY RESULTS: All three species of Pedicularis were pollinated by bumble-bees. It was found that the adaptive accuracy of female function was much higher than that of male function in all three flower species. Although peak pollen deposition corresponded to the optimal location on the pollinator (i.e. the site of stigma contact) for each species, substantial amounts of pollen were scattered over much of the bees' bodies. CONCLUSIONS: The Pedicularis species studied in the eastern Himalayan region did not conform with Grant's 'Pedicularis Model' of mechanical reproductive isolation. The specialized flowers of this diverse group of plants seem unlikely to have increased the potential for reproductive isolation or influenced rates of speciation. It is suggested instead that the extreme species richness of the Pedicularis clade was generated in other ways and that specialized flowers and substantial pollination accuracy evolved as a response to selection generated by the diversity of co-occurring congeners.

Floral isolation in Pedicularis: how do congeners with shared pollinators minimize reproductive interference?

To minimize interspecific pollination, it has been suggested that pollen is placed on different parts of a pollinator's body corresponding to the conspecific location of pollen pickup by the stigma. Although Pedicularis is regarded as a classic example of pollinator-mediated floral isolation, such reciprocal pollen placement has not been demonstrated experimentally. This leads us to question previous observations of pollen release in Pedicularis species. Here, we show that pollen grains are released from the tip, rather than the basal opening, of the galea (the hoodlike upper lip of the corolla) in eight nectarless Pedicularis species, mimicking pollen release from poricidal anthers. We used safranin-stained pollen within anthers to track pollen placement in three Pedicularis species, and showed that pollen was deposited on numerous parts of the bumblebee's body. However, fluorescent powder placed on the stigmas to detect the contact location on the bumblebee's body was deposited mainly on the major position of pollen placement in each of the three species. Such segregation of pollen placement and pickup between species sharing the same pollinator probably helps to reduce reproductive interference, but the positions of pollen placement and stigma contact on the bumblebee's body were not as precise as previously thought.