Novel indices reveal that pollinator exposure to pesticides varies across biological compartments and crop surroundings.

Declines in insect pollinators have been linked to a range of causative factors such as disease, loss of habitats, the quality and availability of food, and exposure to pesticides. Here, we analysed an extensive dataset generated from pesticide screening of foraging insects, pollen-nectar stores/beebread, pollen and ingested nectar across three species of bees collected at 128 European sites set in two types of crop. In this paper, we aimed to (i) derive a new index to summarise key aspects of complex pesticide exposure data and (ii) understand the links between pesticide exposures depicted by the different matrices, bee species and apple orchards versus oilseed rape crops. We found that summary indices were highly correlated with the number of pesticides detected in the related matrix but not with which pesticides were present. Matrices collected from apple orchards generally contained a higher number of pesticides (7.6 pesticides per site) than matrices from sites collected from oilseed rape crops (3.5 pesticides), with fungicides being highly represented in apple crops. A greater number of pesticides were found in pollen-nectar stores/beebread and pollen matrices compared with nectar and bee body matrices. Our results show that for a complete assessment of pollinator pesticide exposure, it is necessary to consider several different exposure routes and multiple species of bees across different agricultural systems.

Is self-incompatibility a reproductive barrier for hybridization in a sympatric species?

PREMISE: Barriers at different reproductive stages contribute to reproductive isolation. Self-incompatibility (SI) systems that prevent self-pollination could also act to control interspecific pollination and contribute to reproductive isolation, preventing hybridization. Here we evaluated whether SI contributes to reproductive isolation among four co-occurring Opuntia species that flower at similar times and may hybridize with each other. METHODS: We assessed whether Opuntia cantabrigiensis, O. robusta, O. streptacantha, and O. tomentosa, were self-compatible and formed hybrid seeds in five manipulation treatments to achieve self-pollination, intraspecific cross-pollination, open pollination (control), interspecific crosses or apomixis, then recorded flowering phenology and synchrony. RESULTS: All species flowered in the spring with a degree of synchrony, so that two pairs of species were predisposed to interspecific pollination (O. cantabrigiensis with O. robusta, O. streptacantha with O. tomentosa). All species had distinct reproductive systems: Opuntia cantabrigiensis is self-incompatible and did not produce hybrid seeds as an interspecific pollen recipient; O. robusta is a dioecious species, which formed a low proportion of hybrid seeds; O. streptacantha and O. tomentosa are self-compatible and produced hybrid seeds. CONCLUSIONS: Opuntia cantabrigiensis had a strong pollen-pistil barrier, likely due to its self-incompatibility. Opuntia robusta, the dioecious species, is an obligate outcrosser and probably partially lost its ability to prevent interspecific pollen germination. Given that the self-compatible species can set hybrid seeds, we conclude that pollen-pistil interaction and high flowering synchrony represent weak barriers; whether reproductive isolation occurs later in their life cycle (e.g., germination or seedling survival) needs to be determined.

Distribution of exchangeable Ca2+ during the process of Larix decidua Mill. pollination and germination.

The involvement of Ca2+ ions in angiosperms sexual processes is well established, while in gymnosperms, such knowledge remains limited and is still a topic of discussion. In this study, we focused on Larix decidua, using Alizarin-red S staining and the pyroantimonate method to examine the tissue and subcellular distribution of free and loosely bound Ca2+ ions at different stages of the male gametophyte's development and its interaction with the ovule. Our findings show that in larch, both the germination of pollen grains and the growth of pollen tubes occur in an environment rich in Ca2+. These ions play a crucial role in the adhesion of the pollen grain to the stigmatic tip and its subsequent movement to the micropylar canal. There is a significant presence of free and loosely bound Ca2+ ions in both the fluid of the micropylar canal and the extracellular matrix of the nucellus. As the pollen tube extends through the nucellus, we observed a notable accumulation of Ca2+ ions just above the entry to the mature archegonium, a region likely crucial for the male gametophyte's directional growth. Meanwhile, the localized presence of free and loosely bound Ca2+ ions within the egg cell cytoplasm may inhibit the pollen tubes growth and rupture, playing an important role in fertilization.

Flower opening dynamics, pollen-ovule ratio, stigma receptivity and stigmatic pollen germination (in-vivo) in Chaenomeles speciosa (Sweet) Nakai.

Although Chaenomeles is widely used in horticulture, traditional Chinese medicine and landscape greening, insufficient research has hindered its breeding and seed selection. This study investigated the floral phenology, floral organ characteristics, palynology, and breeding systems of Chaenomeles speciosa (Sweet) Nakai. The floral characteristics of C. speciosa were observed both visually and stereoscopically. The microstructures of the flower organs were observed using scanning electron microscopy. Pollen stainability was determined using triphenyl tetrazolium chloride staining. Stigma receptivity was determined using the benzidine-H2O2 method and the post-artificial pollination pollen germination method. The breeding system was assessed based on the outcrossing index and pollen-ovule ratio. The flowers of C. speciosa were bisexual with a flowering period from March to April. The flowering periods of single flowers ranged from 8 to 19 d, and those of single plants lasted 18-20 d. The anthers were cylindrical, with the base attached to the filament, and were split longitudinally to release pollen. The flower had five styles, with a connate base. The ovaries had five carpels and five compartments. The inverted ovules were arranged in two rows on the placental axis. The stigma of C. speciosa was dry and had many papillary protrusions. In the early flowering stage (1-2 d of flowering), the pollen exhibited high stainability (up to 84.24%), but all stainability was lost at 7 d of flowering. Storage at - 20 °C effectively delayed pollen inactivation. The stigma receptivity of C. speciosa lasted for approximately 7 days, and the breeding system was classified as outcrossing with partial self-compatibility.

Pollen chemical and mechanical defences restrict host-plant use by bees.

Plants produce an array of chemical and mechanical defences that provide protection against many herbivores and pathogens. Putatively defensive compounds and structures can even occur in floral rewards: for example, the pollen of some plant taxa contains toxic compounds or possesses conspicuous spines. Yet little is known about whether pollen defences restrict host-plant use by bees. In other words, do bees, like other insect herbivores, tolerate the defences of their specific host plants while being harmed by non-host defences? To answer this question, we compared the effects of a chemical defence from Lupinus (Fabaceae) pollen and a putative mechanical defence (pollen spines) from Asteraceae pollen on larval survival of nine bee species in the tribe Osmiini (Megachilidae) varying in their pollen-host use. We found that both types of pollen defences reduce larval survival rate in some bee species. These detrimental effects were, however, mediated by host-plant associations, with bees being more tolerant of the pollen defences of their hosts, relative to the defences of plant taxa exploited by other species. This pattern strongly suggests that bees are adapted to the pollen defences of their hosts, and that host-plant use by bees is constrained by their ability to tolerate such defences.

Pollen viability and longevity in Juniperus taxa native to Slovakia.

Pollen viability, dispersion ability and longevity during deep-freezer storage were studied in three Juniperus taxa distributed in Slovakia. All these characteristics of pollen are closely related to the pollination and/or fertilization success of the junipers in nature. Pollen viability varied considerably between the three populations of J. communis and one population of each, J. sibirica and J. communis var. intermedia. Pollen germination rate ranged between 40.75% and 75.06%, and pollen tube length between 30.32 and 40.41 µm. A clear tendency indicates a higher germination rate of J. communis pollen from lower altitudes and reduced germination of J. sibirica and J. communis var. intermedia pollen from higher altitudes. The dispersion potential of the J. communis pollen during its shedding seems relatively low. In 2021, pollen cloud density was diluted at 68.1% at the 4 m distance from the test shrub, in 2022 of 52.1% at the 17 m distance from the pollen source. A deep-freezer storage of juniper pollen in a double-walled polyethylene bag with silica gel was not efficient enough, as indicated by the drop of pollen germination rate of 31.2% in J. communis and of 79.4% in J. sibirica during a 1-year storage period at - 81 °C.

Convergent evolutionary patterns of heterostyly across angiosperms support the pollination-precision hypothesis.

Since the insights by Charles Darwin, heterostyly, a floral polymorphism with morphs bearing stigmas and anthers at reciprocal heights, has become a model system for the study of natural selection. Based on his archetypal heterostylous flower, including regular symmetry, few stamens and a tube, Darwin hypothesised that heterostyly evolved to promote outcrossing through efficient pollen transfer between morphs involving different areas of a pollinator's body, thus proposing his seminal pollination-precision hypothesis. Here we update the number of heterostylous and other style-length polymorphic taxa to 247 genera belonging to 34 families, notably expanding known cases by 20%. Using phylogenetic and comparative analyses across the angiosperms, we show numerous independent origins of style-length polymorphism associated with actinomorphic, tubular flowers with a low number of sex organs, stamens fused to the corolla, and pollination by long-tongued insects. These associations provide support for the Darwinian pollination-precision hypothesis as a basis for convergent evolution of heterostyly across angiosperms.

High pesticide exposure and risk to bees in pollinator plantings adjacent to conventionally managed blueberry fields.

Wildflower plantings adjacent to agricultural fields provide diverse floral resources and nesting sites for wild bees. However, their proximity to pest control activities in the crop may result in pesticide exposure if pesticides drift into pollinator plantings. To quantify pesticide residues in pollinator plantings, we sampled flowers and soil from pollinator plantings and compared them to samples from unenhanced field margins and crop row middles. At conventionally managed farms, flowers from pollinator plantings had similar exposure profiles to those from unenhanced field margins or crop row middles, with multiple pesticides and high and similar risk quotient (RQ) values (with pollinator planting RQ: 3.9; without pollinator planting RQ: 4.0). Whereas samples from unsprayed sites had significantly lower risk (RQ: 0.005). Soil samples had overall low risk to bees. Additionally, we placed bumble bee colonies (Bombus impatiens) in field margins of crop fields with and without pollinator plantings and measured residues in bee-collected pollen. Pesticide exposure was similar in pollen from sites with or without pollinator plantings, and risk was generally high (with pollinator planting RQ: 0.5; without pollinator planting RQ: 1.1) and not significant between the two field types. Risk was lower at sites where there was no pesticide activity (RQ: 0.3), but again there was no significant difference between management types. The insecticide phosmet, which is used on blueberry farms for control of Drosophila suzukii, accounted for the majority of elevated risk. Additionally, analysis of pollen collected by bumble bees found no significant difference in floral species richness between sites with or without pollinator plantings. Our results suggest that pollinator plantings do not reduce pesticide risk and do not increase pollen diversity collected by B. impatiens, further highlighting the need to reduce exposure through enhanced IPM adoption, drift mitigation, and removal of attractive flowering weeds prior to insecticide applications.

Pollen, anther, stamen, and androecium mimicry.

Floral colours represent a highly diverse communication signal mainly involved in flower visitors' attraction and guidance, but also flower discrimination, filtering non-pollinators and discouraging floral antagonists. The divergent visual systems and colour preferences of flower visitors, as well as the necessity of cues for flower detection and discrimination, foster the diversity of floral colours and colour patterns. Despite the bewildering diversity of floral colour patterns, a recurrent component is a yellow UV-absorbing floral centre, and it is still not clear why this pattern is so frequent in angiosperms. The pollen, anther, stamen, and androecium mimicry (PASAM) hypothesis suggests that the system composed of the flowers possessing such yellow UV-absorbing floral reproductive structures, the flowers displaying central yellow UV-absorbing structures as floral guides, and the pollen-collecting, as well as pollen-eating, flower visitors responding to such signals constitute the world's most speciose mimicry system. In this review, we call the attention of researchers to some hypothetical PASAM systems around the globe, presenting some fascinating examples that illustrate their huge diversity. We will also present new and published data on pollen-eating and pollen-collecting pollinators' responses to PASAM structures supporting the PASAM hypothesis and will discuss how widespread these systems are around the globe. Ultimately, our goal is to promote the idea that PASAM is a plausible first approach to understanding floral colour patterns in angiosperms.

Plant-pollinator network architecture does not impact intraspecific microbiome variability.

Variation in how individuals interact with food resources can directly impact, and be affected by, their microbial interactions due to the potential for transmission. The degree to which this transmission occurs, however, may depend on the structure of forager networks, which determine the community-scale transmission opportunities. In particular, how the community-scale opportunity for transfer balances individual-scale barriers to transmission is unclear. Examining the bee-flower and bee-microbial interactions of over 1000 individual bees, we tested (1) the degree to which individual floral visits predicted microbiome composition and (2) whether plant-bee networks with increased opportunity for microbial transmission homogenized the microbiomes of bees within that network. The pollen community composition carried by bees was associated with microbiome composition at some sites, suggesting that microbial transmission at flowers occurred. Contrary to our predictions, however, microbiome variability did not differ based on transfer opportunity: bee microbiomes in asymmetric networks with high opportunity for microbial transfer were similarly variable compared to microbiomes in networks with more evenly distributed links. These findings suggest that microbial transmission at flowers is frequent enough to be observed at the community level, but that community network structure did not substantially change the dynamics of this transmission, perhaps due to filtering processes in host guts.

Investigating the longstanding mystery of stigma length differences between morphs of distylous plants.

Longer stigmas in short-styled morphs of distylous plants have been considered an adaptive characteristic that increases intermorph pollen deposition. The greater pollen deposition in short-styled stigmas may be a by-product of their longer length, making deposition effectiveness comparison between morphs unfeasible. Thus, investigating which morph has the relatively most efficient stigma (i.e., pollen deposition per unit length) can boost our understanding of the adaptive significance of longer stigmas. Here, we compared pollen deposition between morphs relative to stigma length and assessed whether short-styled stigmas are more, less, or equally effective in receiving pollen grains per unit length. We reviewed the literature to characterize the extent of sigma length differences between morphs using the two most speciose distylous genera as model systems: Palicourea and Psychotria (Rubiaceae). Then, we conducted a between-morph comparison of raw and relative pollen depositions in a Palicourea rigida population. We confirmed that short-styled stigmas were longer than long-styled stigmas in both Palicourea (194.31% longer) and Psychotria (52.59% longer) flowers. Furthermore, in the focal Pal. rigida, although short-styled stigmas were 268.04% longer and received 97.04% more pollen grains than long-styled stigmas, the relative pollen deposition had a reverse pattern, with short-styled stigmas being two-times less efficient in receiving pollen. Our results indicate that the longer length of the short-styled stigmas may have a compensatory effect, increasing the chances of pollen grains reaching the stigma and probably maintaining disassortative intermorph pollination in distylous plants.

Effective population size mediates the impact of pollination services on pollen limitation.

Inadequate pollen receipt limits flowering plant reproduction worldwide. Ecological causes of pollen limitation (PL), like pollinator scarcity and low plant abundance, have been a primary research focus. The genetic diversity of plant populations could impact both quantity and quality components of PL in concert with ecological factors, yet empirical examples are lacking. We evaluated joint effects of ecological factors (flower abundance, pollinator visitation) and genetic effective population size (NE) on PL across 13 populations of the common herb Argentina anserina. We used a histological approach with 5504 styles from 1137 flowers to separate quantity and quality components of PL, and link these to reproductive output. NE and pollinator visitation interacted to shape PL, but NE had stronger direct effects. Effectively smaller populations experienced stronger quantity PL, and controlled crosses in a pollinator-free environment revealed that stigmatic pollen quantity was an intrinsic population-level attribute that increased with NE. Pollinator visitation enhanced pollen quality, but only in effectively larger populations. Quantity and quality PL negatively impacted fruit and seed set, respectively. Results highlight that PL is dictated by plant population genetic diversity in addition to commonly evaluated ecological factors. Efforts to support pollinators will more strongly enhance plant reproduction in genetically diverse populations.

Evolution of flowering time due to variation in the onset of pollen dispersal among individuals.

The evolution of flowering time is often attributed to variations in pollinator rates over time. This study proposes that flowering time can evolve through siring success variation among individuals caused by differential pollen dispersal timing (a result of flowering time variation). By building quantitative genetic models, I show that flowering time evolves to be earlier when the pollen removal rate is low and pollen deposition rate is high, and the fertilization ability of removed pollen declines slowly. Using evolutionary game theory, I show that the evolutionarily stable variance of flowering time is large when the pollen removal rate is either low or high, the pollen deposition rate is moderate, and the fertilization ability of removed pollen declines rapidly. Investigation of the coevolution of flower longevity and flowering time shows that under constant pollination rates, late flowering will be correlated with long-lived flowers due to nonrandom mating, which suggests that the observed correlation between late flowering and short-lived flowers is caused by other factors, such as declining pollination rates during late-stage flowering. I discuss how altered pollination rates under climate change will influence flowering time evolution and the importance of distinguishing between pollen removal and deposition rates.

150 Years of Coevolution Research: Evolution and Ecology of Yucca Moths (Prodoxidae) and Their Hosts.

Yucca moths (Tegeticula and Parategeticula) are specialized pollinators of yucca plants, possessing unique, tentacle-like mouthparts used to actively collect pollen and deposit it onto the flowers of their hosts. The moths' larvae feed on the developing seeds and fruit tissue. First described in 1873, the yucca-yucca moth pollination system is now considered the archetypical example of a coevolved intimate mutualism. Research conducted over the past three decades has transformed our understanding of yucca moth diversity and host plant interactions. We summarize the current understanding of the diversity, ecology, and evolution of this group, review evidence for coevolution of the insects and their hosts, and describe how the nature of the interaction varies across evolutionary time and ecological contexts. Finally, we identify unresolved questions and areas for future research.

Linking pollen limitation and seed dispersal effectiveness.

Seed production and dispersal are crucial ecological processes impacting plant demography, species distributions and community assembly. Plant-animal interactions commonly mediate both seed production and seed dispersal, but current research often examines pollination and seed dispersal separately, which hinders our understanding of how pollination services affect downstream dispersal services. To fill this gap, we propose a conceptual framework exploring how pollen limitation can impact the effectiveness of seed dispersal for endozoochorous and myrmecochorous plant species. We summarize the quantitative and qualitative effects of pollen limitation on plant reproduction and use Optimal Foraging Theory to predict its impact on the foraging behaviour of seed dispersers. In doing so, we offer a new framework that poses numerous hypotheses and empirical tests to investigate links between pollen limitation and seed dispersal effectiveness and, consequently, post-dispersal ecological processes occurring at different levels of biological organization. Finally, considering the importance of pollination and seed dispersal outcomes to plant eco-evolutionary dynamics, we discussed the implications of our framework for future studies exploring the demographic and evolutionary impacts of pollen limitation for animal-dispersed plants.

Supplementing honey bee (Hymenoptera: Apidae) colonies with pollen increases their pollinating activity on nectariferous crops with anthers isolated from stigmas.

The western honey bee (Apis mellifera L.) is the most globally used managed pollinator species, but it can have limited pollinating activity on nectariferous crops displaying anthers isolated from stigmas, i.e., when anthers are spatially or temporally separated from stigma within or between flowers. We supplemented honey bee colonies with pollen in the combs or in paste form laid on top of the hive frames to test if these treatments could reduce their pollen foraging and increase their pollinating activity in a monoecious and nectariferous cultivar of cantaloupe melon (Cucumis melo L.), in comparison with control colonies not supplemented. We recorded the pollen forager density per flower, the number of pollen grains deposited per stigma and their resulting fruit set, seed set and fruit mass, before and after the colony pollen supplementations. The number of pollen grains deposited by honey bees on stigmas increased gradually after pollen supplementation in the combs. But pollen foraging decreased only moderately, and no effect could be observed on any yield component except the seed set. On the other hand, there was no effect of the pollen paste laid on top of the frames either on stigmatic pollen loads, on colony pollen foraging or on any yield component. Supplementing honey bee colonies with pollen in the combs can therefore be an effective means for increasing their pollinating activity in nectariferous crops displaying anthers isolated from stigmas, e.g., Amaryllidaceae, Apiaceae, Cucurbitaceae, avocado, all hybrid seed productions. The context for the potential use of pollen substitutes is discussed.

Microbes, the 'silent third partners' of bee-angiosperm mutualisms.

While bee-angiosperm mutualisms are widely recognized as foundational partnerships that have shaped the diversity and structure of terrestrial ecosystems, these ancient mutualisms have been underpinned by 'silent third partners': microbes. Here, we propose reframing the canonical bee-angiosperm partnership as a three-way mutualism between bees, microbes, and angiosperms. This new conceptualization casts microbes as active symbionts, processing and protecting pollen-nectar provisions, consolidating nutrients for bee larvae, enhancing floral attractancy, facilitating plant fertilization, and defending bees and plants from pathogens. In exchange, bees and angiosperms provide their microbial associates with food, shelter, and transportation. Such microbial communities represent co-equal partners in tripartite mutualisms with bees and angiosperms, facilitating one of the most important ecological partnerships on land.

Incomplete resupination during floral development leads to pollination failure in a slipper orchid.

In many families, plants undergo floral resupination by twisting through approximately 180° during floral development so that the flower is effectively positioned upside down. In most orchids, resupination results in the median petal (i.e., the labellum) becoming lowermost, which plays a crucial role in pollination by serving as a landing platform or as a trapping device, or both. Incomplete resupination is predicted to lead to reduced pollination, although tests of this assumption are still lacking. We investigated the effect of resupination using Phragmipedium vittatum, a rare lady's slipper orchid whose specialized labellum forms a trapping device. First, we surveyed the natural occurrence of incomplete resupination. Then we manipulated flowers into non- (≈0°), half- (≈90°), and fully resupinate (≈180°) positions to test the effect of orientation on pollen smear removal and deposition by pollinators (female hoverflies). We found that ca. 10% of flowers in the natural population were not fully resupinate, being either non- (upward, 0-60°) or half-resupinate (sideward, 60-120°). The change in orientation prevented the effectiveness of pollination by hoverflies since no pollen smear removal or deposition were found in flowers from non- and half-resupinate treatments. Although these flowers still attracted hoverflies, they were not trapped effectively. As this orchid is incapable of autonomous self-pollination, flowers that do not resupinate fail to set fruits. These results highlight the importance of correct floral orientation provided by resupination to ensure pollination in orchids and other resupinate flowers.

Gametophytic self-incompatibility in Maxillariinae orchids.

Gametophytic self-incompatibility (GSI) has been mainly described in species-rich clades within Orchidaceae. We report GSI for a genus within Maxillariinae, one of the most conspicuous and diverse subtribes of neotropical orchids. Here, we describe the reproductive system of Brasiliorchis picta, B. phoenicanthera, and B. porphyrostele. Anatomical studies of fruit development showed that pollen tubes of aborted, self-pollinated flowers degenerate half-way in the stylar channel and never reach the ovules. Spontaneous self-pollination and emasculation set no fruits for none of the sampled species, thus supporting the hypothesis that these three species studied rely on the agency of pollinators and pollinator-mediated cross-pollination to set fruit. Fruit set from cross-pollinations ranged from 33.4 to 77.5%. One self-pollinated fruit of B. porphyrostele developed to completion. All other fruits aborted between 10 and 21 days after pollination. These data support previous evidence of variable strength GSI being exhibited in orchid species. Additional studies of self-incompatibility systems are needed to evaluate their role in species diversification and evolution of reproductive strategies in Maxillariinae and to allow for effective conservation strategies of threatened orchid species.

Darwin's inflorescence syndrome is indeed associated with bee pollination.

KEY MESSAGE: A relationship between vertical acropetal inflorescences with protandrous flowers and bee pollination was hypothesized by Darwin back in 1877. Here we provide empirical evidence supporting this association across the angiosperms. Plant reproduction is not only determined by flower traits but also by the arrangement of flowers within inflorescences. Based on his observations of the orchid Spiranthes autumnalis, Darwin proposed in 1877 that bee-pollinated plants presenting protandrous flowers on vertical acropetal inflorescences, where proximal flowers open first, can exploit the stereotypical foraging behavior of their pollinators (i.e., upward movement through the inflorescence) to promote pollen exportation and reduce self-pollination. In these inflorescences, male-phase flowers lie spatially above female-phase flowers. To examine this untested hypothesis, we compiled literature information from 718 angiosperms species and evaluated the association between vertical acropetal inflorescences with protandrous flowers and bee pollination within a phylogenetic comparative framework. Results reveal that this type of inflorescence is indeed more common in species pollinated by bees. Moreover, this association does not seem to be weakened by the presence of alternative self-pollination avoidance mechanisms, like self-incompatibility, suggesting that this inflorescence type benefits mainly male rather than female fitness. Other inflorescence types placing male-phase flowers above female-phase flowers, e.g., vertical basipetal inflorescences with protogynous flowers, do not provide strong evidence of a differential association with pollination by bees. Female-biased nectar production in vertical acropetal inflorescences with protandrous flowers may reinforce the behavior of bees to fly upwards, rendering Darwin's configuration more adaptive than other inflorescence configurations.