Heterospecificity of body pollen varies across and within functional groups of pollinators

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To capture presumed similarities in these selective forces, pollinators have traditionally been organized into functional groups, often corresponding to taxonomic classifications. However, given the likely diversity of foraging behavior among pollinators, groupings based solely on taxonomy or morphology may not adequately reflect variation in pollen transfer efficiency, particularly in terms of heterospecific pollen transfer (HPT) potential. To explore this possibility, we assessed how 11 groups of insect pollinators in a Japanese satoyama landscape, classified by both taxonomy and putative behavioral differences, varied in the heterospecificity of pollen on their bodies. Despite being collected from flowers of the same Ligustrum trees in close proximity, the body pollen profiles of these insects significantly differed—both among and within the 11 groups—in terms of the proportion of heterospecific (non- Ligustrum ) pollen, the composition of pollen species, and the diversity of pollen species. Notably, marked variations were found within bees, flies, and beetles, as well as between males and females of the same bumble-bee species. Based on these data, we discuss how the observed variations could offer new insights into conventional views on functional groups of pollinators, which are largely based on taxonomy or morphological/size similarities. Body pollen Functional groups Heterospecific pollen transfer Pollen species diversity Plant-animal interactions Pollinators Figures Figure 1 Figure 2 Figure 3 Introduction Selection pressures on floral phenotypes exerted by distinct types of pollinators are widely regarded as key drivers of floral diversification and convergence. To capture presumed similarities in these selective forces, pollinators have traditionally been grouped into functional groups (Vogel 1954 ; Faegri and van der Pijl 1979; Fenster et al. 2004 ), which often corresponded to taxonomic classifications such as bees, flies, butterflies, moths, beetles, birds, and bats (Fenster et al. 2004 ). This taxonomically oriented framework likely stems from the assumption that a pollinator's contribution to plant reproduction is primarily determined by the amount of pollen it removes from anthers and deposits onto stigmas of other plants (Bierzychudek 1981; Ne'eman et al. 2010; Thomson et al. 2000 ; Johnson and Harder 2023 ), and that animals with similar sizes and morphologies—often (though not always) shared within taxonomic groups—therefore exhibit comparable levels of pollen transport and, by extension, exert similar selective pressures on flowers (Fenster et al 2004 ). While this approach has some practical utility, it may overlook important differences in the quality of pollen transfer among pollinators, which are strongly influenced by their foraging behavior among flowers and plants. For example, studies have shown that pollinators can differ markedly in the proportion of self- versus outcross pollen on their bodies (Hasegawa et al. 2015 ) and in the genetic diversity of pollen donors they transfer (Valverde et al. 2019 ). In addition to these genetic components, accumulating evidence suggests that a pollinator’s contribution also depends on how frequently it moves between plant species, as this increases the likelihood of heterospecific pollen transfer (HPT)—a process that can reduce male fitness by diverting pollen to heterospecific stigmas (Muchhala and Thomson 2012 ), and female fitness through mechanisms such as stigma clogging (Galen and Gregory 1989 ), stigma closure (Waser and Fugate 1986 ), allelopathic or physical interference with pollen germination and growth (Buchman and Campbell 2016 ; Murphy 2000 ), or ovule usurpation and hybridization (Harder et al. 1993; Burgess et al. 2008 ). The propensity to induce HPT can vary among pollinators, depending on various factors, including their perceptual and cognitive abilities (Latty and Trueblood 2020 ), dietary breadth (Brosi 2016 ), flower constancy (Goulson and Wright 1998 ; Chittka et al. 1999; Amaya-Márquez 2009), foraging range (Grüter and Hayes 2022 ), sampling frequency (Heinrich 1979 ; Dunlap et al. 2017 ), pollen foraging behavior (Russell et al. 2021 ), among others. Some suggestive observations have been made previously in this regard. For example, butterflies have been observed flying longer distances than other flower visitors, often bypassing nearby plants (Schmitt 1980 ; Herrera 1987 ), potentially increasing the likelihood of cross-species pollen transfer (Alarcón 2010 ; Zhao et al. 2019 ). A study on 85 bee taxa found that eusocial bees exhibit 67.5% greater pollen diet diversity compared to noneusocial bees (Devkota et al. 2024 ), indicating that they are inferior pollinators in terms of the heterospecificity of body pollen. Moreover, varying tendencies toward HPT may exist even within conventionally recognized functional groups of pollinators. For example, male bumble bees have been observed to collect pollen from a narrower range of flowers than workers (Wolf and Moritz 2014 ). Despite these indications, our current knowledge is still limited and fragmented regarding how the propensity to cause HPT varies among and within different groups of pollinators. In order to gain further insight into this issue, we conducted a field survey in a Japanese satoyama landscape to quantify and compare the composition of body pollen carried by a diverse range of insects visiting flowers of border privet, Ligustrum obtusifolium Siebold et Zucc. (Oleaceae), and to assess their potential to induce HPT. Ligustrum obtusifolium is a generalist flowering species that attracts a broad range of insect pollinators. We therefore assumed that visitors collected from a few nearby L. obtusifolium plants would share overlapping foraging ranges and, to some extent, access to a common pool of co-flowering species. This assumption allowed us to quantitatively compare their inherent propensity to forage across multiple species—a comparison that would have been less straightforward if insects had been sampled from different plant species or separate locations. We examined the composition of pollen species found on each pollinator's body, and compared (1) the proportion of heterospecific (non- Ligustrum ) pollen, (2) the composition of pollen species, and (3) the diversity of pollen species. Drawing from these data, we discuss how the observed variations in body pollen heterospecificity, or the propensity to induce HPT, could offer new insights into conventional views on functional groups of pollinators, which are largely based on taxonomy or morphological/size similarities. Materials and methods Insect sampling Ligustrum obtusifolium is a small deciduous shrub or tree that can reach a height of 3–6 m. In early summer, it produces hermaphroditic flowers that are arranged in dense inflorescences. Each flower has a tubular corolla (10 mm in length, 5 mm in diameter) with four lobes, typically white or cream-colored. Its nectar and a sweet odor strongly attract insect visitors of various taxa, including bees, wasps, flies, beetles, butterflies, and nocturnal moths (Yokoi et al., 2008 ; Ikenoue and Kanai, 2010 ). Our preliminary survey at this site in 2019 identified 125 insect species from six orders, 37 families, and 109 genera (Table S1). This pollinator-generalist flower often serves as the hub species within local pollination networks, making it suitable for the purpose of this study. We conducted the study in Susomi no Mori (meaning 'foothill forest'), a small secondary forest at the foot of Mt. Tsukuba, Ibaraki, Japan (36°11'34.1" N, 140°06'44.6" E), surrounded by traditional farmland, including rice paddies. This forest has been managed by the NPO Tsukuba Environmental Forum since 2006 to conserve its satoyama landscape and sustain local cultural heritage. From 24 to 31 May 2020, we conducted around-the-clock sampling of flower visitors on three closely growing L. obtusifolium plants. We observed these trees during the day (for a total of 30 hours) and the night (for a total of 8 hours), capturing all the flower visitors using an insect net. At least 25 individuals were collected for each of the 11 insect groups described in the following. Nocturnal observations were made using a headlamp of > 350 lumens (LEDLenser® H14R, Solingen, Germany) covered with red cellophane. We placed the captured insects separately in centrifuge tubes of appropriate sizes, ranging from 1.5 ml to 30 ml, or triangular paper envelopes for lepidopterans. We then transported them back to the laboratory in a cooler box and preserved them in a freezer set to -20°C. The sampled insects were classified into 11 groups based on their distinct behavior and morphology: worker bumble bees ( Bombus ardens ), male bumble bees ( B. ardens ), mining bees ( Andrena spp.), the other bees, wasps, hairy scarab beetles ( Oxycetonia jucunda ), the other beetles, hoverflies (Syrphidae), the other flies, butterflies, and nocturnal moths. Bumble bees are grouped separately from other bees due to their high cognitive abilities, which are associated with efficient tactics for locating and obtaining resources, such as individual specialization (Heinrich 1976a ) and memory-based spatial foraging (Cartar 2004 , Ohashi and Thomson 2009 , Ogilvie and Thomson 2016 ). Taking advantage of their highly frequent visits, we further separated them into males and workers (females) to see if there were differences derived from their foraging requirements (Wolf and Moritz 2014 ). Among other bees, we also treated mining bees ( Andrena spp.) as a distinct group because this genus comprises numerous oligolectic species (Danforth 2007 ). One abundant scarab species, O. jucunda , was distinguished from other coleopterans based on its distinctly dense hair and strong flight capabilities. For dipterans, we separated hoverflies from other (muscoid and oestroid) flies because of their strong dependence on floral resources and their selective foraging (Larson et al. 2001 ; Inouye et al. 2015 ). Finally, butterflies and nocturnal moths were distinguished based on their activity time, reliance on different sensory modalities (i.e., vision and olfaction) (Brantjes 1978 ; Cunningham et al. 2004 ; Balkenius et al. 2006 ), and the resulting expectations regarding their foraging repertoires. Note that nocturnal moths on L. obtusifolium were all settling moths, which land on flowers to probe nectar. No hawkmoths (Sphingidae) were observed visiting flowers of L. obtusifolium . Diurnal moths were excluded from the analysis due to the small sample size. Pollen identification and counting Pollen grains were collected from insect bodies following the method described by Nikkeshi et al. ( 2016 ), i.e., by rinsing the insect body with a sucrose solution. We carefully avoided any contamination with pollen that could not be used for pollination, by excluding samples from insects that had regurgitated or excreted pollen, as well as those from female bees with visibly distinct sizes of pollen loads on their corbiculae (less than 10% of the collected individuals). Each insect was placed in an appropriate size centrifuge tube (1.5–3.0 ml) with a 0.4 mol/l sucrose solution. After shaking the tube up and down by hand, being careful not to destroy the insect body, we agitated the tube for 1 minute on a vortex mixer (Tube Mixer Trio HM-1F, AS ONE). Next, we sonicated the tube for 5 minutes in an ultrasonic bath (UT-106H, SHARP). Finally, we agitated the tube again on a vortex for 1 minute, after which we shook the tube once by hand. This method has been shown to separate 96.5% or more of the total pollen attached to the body surface of an insect (Nikkeshi et al. 2016 ). For each insect, we prepared five microscope slides, each of which was made from 10 µl of pollen suspension. We then examined the slides under a microscope (OLYMPUS CX43) equipped with a microscope camera (AdvanVision AdvanCam-HD2s) to identify and count pollen grains. Pollen identification was performed by referring to a collection of pollen slides and microscopic images of flowering species around our study site. Before and during the flowering season of L. obtusifolium (15–31 May 2020), we conducted an exhaustive search by foot for co-flowering species within a 1-km radius of the L. obtusifolium trees. Each flowering species was identified, and the coordinates of the individual closest to our focal L. obtusifolium trees were recorded using a handheld GPS receiver (model: eTrex 30x; Garmin, Olathe, KS, USA) with an accuracy of less than two meters (Table S2). The anthers were collected from these flowers just before/after anthesis and placed in microtubes (1.5 ml) with forceps. In the laboratory, we stored the anthers in a dried condition by opening the lids of microtubes and placing them in a sealed container with calcium chloride. Similarly to the collection of insect body pollen described above, the pollen in these anthers was dissolved in 1 ml of a 0.4 mol/l sucrose solution by agitating for 1 minute on a vortex mixer, followed by 5 minutes of sonication in an ultrasonic bath. We prepared a microscopic slide from 10 µl of each pollen suspension and captured images using the same microscope camera system. After counting the pollen on five slides for each insect, the total number of pollen grains of the i-th plant species, NP i , was estimated as follows: NP i = (No. of pollen grains of the i-th species in a 50-µl suspension) x (Total volume of the pollen suspension in microliter unit) / 50 (1). Using the above method, Nikkeshi et al. ( 2021 ) have shown that 95% or more of the pollen species on the body surface of a single insect can be detected. Statistical analysis To contrast and compare the potential impact of each pollinator group on HPT, we focused on three aspects of heterospecificity of insect body pollen, i.e., i) proportion of heterospecific pollen (= pollen other than L. obtusifolium ), ii) pollen species composition, and iii) pollen species diversity, as described in more detail below. All analyses were performed using R (R Core Team 2021 ), and all graphs were created using the ggplot2 package (Wickham 2016 ). First, we determined whether the proportion of heterospecific pollen differed among pollinator groups visiting L. obtusifolilum by fitting a generalized linear-mixed model (GLMM) with a logit link function and a binomial error distribution. The proportion of heterospecific pollen, calculated as the number of heterospecific pollen grains other than L. obtusifolium devided by the total number of pollen grains on a insect's body (∑NP i ), was used as the response variable. We considered pollinator group as a fixed effect and individual insects as a random effect. For the model fitting, the calculation of marginal and conditional R 2 (Nakagawa and Schielzeth 2013 ), the type-II Wald chi-square tests, and the estimation of marginal (model-adjusted) means and 95% confidence intervals with the comparison of means, we used glmer in ‘lme4’ (Bates et al. 2014), r.squaredGLMM in ‘MuMIn’ (Barton and Barton 2015 ), Anova in ‘car’ (Fox et al. 2007 ), and emmeans with pairs in ‘emmeans’ (Searle et al. 1980 ; Lenth et al. 2020), respectively. Second, we investigated whether and to what extent the species composition of body pollen varied among different pollinator groups. To obtain a dissimilarity matrix reflecting the compositional dissimilarity among pollinator groups, we applied the Bray-Curtis distance to the pollen species matrix using the vegdist in ‘vegan’ (Oksanen et al., 2022 ). Next, we performed a non-metric multidimensional scaling (NMDS, Kruskal 1964 ) using metaMDS function in ‘vegan’ to reduce the dimensionality of species composition to two while minimizing the departure from the distance relationships in the original data. Finally, we performed a PERMANOVA to test whether pollen species composition differed among pollinator groups using adonis function in ‘vegan’. Finally, we assessed the diversity of pollen species on each insect's body, as insects with a more diverse body pollen may deposit more foreign pollen onto the stigmas. The diversity of body pollen species was evaluated with Shannon-Wiener Index (Shannon 1948 ). Biodiversity generally reflects the two often independent components, i.e., richness (number of species) and evenness (equitability of the proportional abundances of those species) (Sheldon 1969 ; Hurlbert 1971 ; Soininen et al. 2012 ; Tuomisto 2012 ). Therefore, we compared the characteristics of the observed species diversity of body pollen among 11 pollinator groups by plotting the data on a two-dimensional space with two orthogonal axes representing richness and evenness. We measured evenness using the Sheldon's equitability index (Sheldon 1969 ) calculated as: E = e H' /S (2), where e is the base of the natural logarithms, H' is the Shannon-Wiener index, and S is pollen species richness on the insect's body. The quantity e H' is the minimum number of equally common species that could yield the observed diversity H'. To correct for differences in the amount of body pollen among insects, we computed e H' and S based on rarefaction for a sample size of 10 pollen grains, using iNEXT function in ‘iNEXT’ (Hsieh et al. 2016 ). Results We examined 331 insects and recorded between 10 and 1,122,500 pollen grains on their body surfaces. The pollen grains found on an insect’s body contained up to eight plant species. The number of body pollen grains per individual in each insect group is summarized in Table 1. The species profile of body pollen across 11 insect groups is shown in Fig. S1. The following sections present analyses focusing on three aspects of body pollen heterospecificity. Proportion of heterospecific pollen We observed a significant variation in the proportion of heterospecific (non- Ligustrum ) pollen among the 11 observed insect groups (χ 2 = 146.5, P < 0.001, Type II Wald chi-square test; Fig. 1 ). The variation in the proportion of heterospecific pollen among individuals was explained by the insect group, accounting for 29% of the variation, while the majority of the remaining variation was attributed to a random effect, namely, insect individuals (marginal R 2 = 0.29, 0.99 < conditional R 2 < 1; Nakagawa & Schielzeth’s pseudo-R 2 for GLMM). The proportion of heterospecific pollen was particularly high in mining bees and hairy scarab beetles. In contrast, bumble bees exhibited much lower heterospecificity, with workers showing significantly higher values than males (Benjamini-Hochberg adjusted P < 0.01). We also observed significant variation in heterospecific pollen proportion within the same order or superfamily. Specifically, mining bees, consisting of three Andrena species (Andrenidae), had a significantly higher proportion of heterospecific pollen than bumble bees ( B. ardens ) and the 'other bees' group (13 species from Apidae, Megachilidae, Halictidae, and Colletidae) (Benjamini-Hochberg adjusted P < 0.0001). Interestingly, male bumble bees showed significantly lower heterospecificity than workers (male: N = 30, marginal mean ± 95% CI = 0.04 ± 0.06; worker: N = 30, 0.16 ± 0.19). Hairy scarab beetles ( O. jucunda ) exhibited a significantly higher proportion of heterospecific pollen than the 'other beetles' group (13 species from Scarabaeidae, Cerambycidae, Elateridae, Cantharidae, Nitidulidae, Melyridae, Oedemeridae, and Carabidae) (Benjamini-Hochberg adjusted P < 0.0001). Although male hairy scarab beetles showed slightly lower heterospecificity than females (male: N = 13, marginal mean ± 95% CI = 0.64 ± 0.29; female: N = 17, 0.85 ± 0.14), this trend was not statistically significant, likely due to small sample sizes (P > 0.17, Type II Wald chi-square test). On the other hand, the difference in heterospecificity of body pollen was not significant between the two Diptera groups, i.e., hoverflies (nine species from Syrphidae) and the 'other flies' (8 species from Muscidae, Calliphoridae, Anthomyiidae, and Conopidae) (Benjamini-Hochberg adjusted P = 0.070). Likewise, we found no significant difference between butterflies (nine species from Papilionidae, Pieridae, Nymphalidae, and Hesperiidae), and nocturnal moths (17 species from Crambidae, Geometridae, and Noctuidae) (Benjamini-Hochberg adjusted P = 0.26). Pollen species composition Pollen species composition varied significantly among the 11 observed insect groups (P < 0.001, PERMANOVA; Fig. 2 ). This partially reflects the pollen compositional similarities within each insect group. Of particular note, both worker and male bumble bees exhibited highly converged profiles of pollen species among individuals, as demonstrated by the narrow polygonal areas shown in Fig. 2 . The level of similarity among individuals in these groups was more pronounced than in hairy scarab beetles, which also consisted of a single insect species. Close similarity within the same group was not always observed, however. Hoverflies and the 'other flies' showed noticeable within-group variations (Fig. 2 ). Because each group includes nine and eight species, respectively, a significant portion of this variability is likely attributed to interspecific differences. Similar trends were also found in the two non- Bombus bee groups (Fig. 2 ). It should be noted that considerable differences in pollen species composition were observed even among closely related insect groups. The compositional difference between the two Diptera groups, i.e., hoverflies and the 'other flies', as well as between the two bee groups, i.e., mining bees and the 'other bees', falls into this category (Fig. 2 ). Yet another example is the two groups of Lepidoptera, i.e., butterflies and nocturnal moths (Fig. 2 ). This difference reflects that, except for Ligustrum pollen, butterflies often carried a large amount of pollen from a single species of Asteraceae (64% of heterospecific pollen), while nocturnal moths often carried a large amount of pollen from a single species of Citrus (91% of heterospecific pollen) (see Fig. S1j, k for more details). Pollen species diversity Figure 3 shows the relationship between the richness and evenness of body pollen species across 11 insect groups. Consistent with the other two analyses, male bumble bees exhibited the lowest species diversity of body pollen (H' = 0.33). Worker bumble bees were not the second lowest in terms of pollen species diversity (H' = 0.63) because their relatively high richness counteracted the low evenness caused by their highly biased pollen species composition. In contrast to bumble bees, the other two bee groups, i.e., mining bees and the 'other bees', both exhibited the highest levels of pollen species diversity (mining bees: H' = 0.94, other bees: H' = 0.90). All four groups of bees carried several pollen species on their bodies, but in bumble bees, species abundance was highly skewed toward one or two species, whereas in non- Bombus bees, pollen species were more evenly distributed (see also Fig. S1a-d). For another hymenopteran group, wasps, pollen species diversity was intermediate (H' = 0.73). Among the hymenopterans, this group had the lowest pollen richness but the highest evenness. Patterns of pollen species diversity varied within the other insect orders as well. Among beetles, hairy scarab beetles exhibited higher diversity than the 'other beetles' (hairy scarab beetles: H' = 0.74, 'other beetles': H' = 0.55), primarily due to differences in species richness (see also Fig. S1f, g). A similar contrast was found within Lepidoptera, where butterflies exhibited one of the highest levels of pollen species diversity (H' = 0.93) among all insect groups, whereas nocturnal moths showed lower diversity (H' = 0.64), at a similar level to that of worker bumble bees. In contrast to these patterns, we observed little variation in pollen species diversity between the two dipteran groups, with hoverflies (H' = 0.77) and 'other flies' (H' = 0.80) exhibiting similar, intermediate levels. This uniformity in diversity sets Diptera apart from the other insect orders analyzed, despite variations in their pollen-carrying capacities (Table 1) and pollen species composition (Fig. 2 ). Discussion Our study revealed that the 11 insect groups visiting L. obtusifolium flowers differ significantly in all three aspects: the proportion of heterospecific pollen, pollen species composition, and pollen species diversity. Substantial differences were observed not only among phylogenetically distant taxa but also among closely related taxa and even between sexes of the same species. These findings support our initial hypothesis that groupings of pollinators based primarily on taxonomy or morphological and size similarities may not adequately reflect their pollination quality in terms of HPT potential—a factor that may have largely been overlooked in the literature—and reinforce the importance of considering the behavioral aspects of pollinators when defining their functional groups (Maubecin et al. 2021 ). Below, we discuss the major trends in the three-dimensional heterospecificity of body pollen, both among and within conventional functional groups, as well as their possible consequences for plant reproduction. The body pollen of bumble bees exhibited low species diversity and little individual variation (Figs. 2 and 3 ), being predominantly composed of L. obtusifolium and a few other species (Figs. 1 and S1a, b). This pattern was particularly pronounced in males, whose pollen species composition was far more uniform than that of workers (Fig. 3 ). Such extreme pollen constancy suggests that bumble bees not only serve as one of the insect groups carrying the largest amounts of pollen (Table 1) but also contribute little to heterospecific pollen transfer (HPT), both for L. obtusifolium and for co-flowering plant species. This finding may be explained by individual bumble bees' tendency to develop foraging preferences for one or a few species (Heinrich 1976a ), their site fidelity to small areas (Thomson 1996 ; Cartar 2004 ; Ogilvie and Thomson 2016 ), or their flower constancy—where they make consecutive visits to the same flower species while disregarding others, especially when that species forms large clusters (Chittka et al. 1999; Ishii 2006 ; Takagi and Ohashi 2025 ). In particular, the significantly lower pollen species diversity in males compared to workers (Figs. 2 and 3 ) may correspond to their spatial foraging patterns. Ogilvie and Thomson ( 2015 ) observed that male bumble bees tend to forage within smaller, more localized areas than workers, which could contribute to their extreme pollen uniformity. This explanation seems plausible, considering that our L. obtusifolium trees produced enough flowers to saturate individual foragers. In contrast, the body pollen of mining bees ( Andrena spp.), which had the highest per-insect pollen load (Table 1), exhibited the greatest species diversity among the 11 insect groups (Fig. 3 ) and was often dominated by non- Ligustrum species (Figs. 1 and S1c). This finding contradicts the common perception of the family Andrenidae as comprising numerous oligolectic species (Danforth 2007 ), as well as the recognized pattern that solitary bees exhibit lower pollen diet diversity than eusocial bees (Devkota et al. 2024 ). However, it may suggest that all the species found on L. obtusifolium were polylectic—a trait that have evolved later in this lineage (Larkin et al. 2008 ). Similar trends in body pollen were observed for the 'other bees' group, which showed higher species diversity than bumble bees (Fig. 3 ), although Ligustrum pollen accounted for more than 50% of the total (Fig. 1 ). Note that both of these bee groups also exhibited greater individual variation in pollen species composition than bumble bees (Fig. 2 ), although the current data do not rule out the possibility that this variation is largely due to interspecific or interfamily differences. The low individual variation observed in bumble bees, despite their generalist foraging nature (Watts et al. 2016 ; Russell et al. 2017 ; Peat et al. 2005 ), may be partly due to the abundant floral resources of L. obtusifolium , which could have reduced the need for resource partitioning among individuals (Heinrich 1976b ). However, since the other bee pollinators were exposed to the same floral environment, it is likely that these solitary bee species inherently exhibit greater individual variation than bumble bees. Compared to bees, wasps carried fewer pollen species on their bodies (Fig. 3 ) and tended to transport less pollen overall (Table 1). Their pollen-carrying characteristics more closely resembled those of the 'other flies' group rather than the three bee groups. This reduced pollen load may be, in part, due to their lower body hair density and their greater reliance on carnivory over floral resource use. Additionally, the species composition of wasp body pollen was distinct from that of all other insect groups, often being dominated by arrowwood ( Viburnum dilatatum ) (Fig. S1e). This pattern may reflect wasps' preference for dish-shaped flowers with exposed nectaries, such as those of arrowwood, due to their relatively short mouthparts (Fægri and van der Pijl, 1979 ). In contrast to this cross-taxon similarity, the two dipteran groups—hoverflies and the 'other flies'—exhibited distinct body pollen profiles in both total pollen amount (Table 1) and species composition (Figs. 2 and S1h, i). Notably, hoverflies carried five times more pollen than the other flies (Table 1). These differences may reflect a higher floral dependence and visitation frequency in hoverflies compared to other flies. However, these differences do not necessarily indicate that one group is more likely to cause HPT than the other, as they were similar both in the proportion of Ligustrum pollen and pollen species diversity (Figs. 1 and 3 ). Hoverflies may be considered a distinct functional group—separate from other flies—and may provide more effective pollination without increasing the risk of HPT, particularly in plant species where HPT is not a major concern. Hairy scarab beetles ( O. jucunda ) stood out from the 'other beetles' group with a lower proportion of Ligustrum pollen (Fig. 1 ), a distinct pollen species profile (Fig. 2 ), higher species diversity (Fig. 3 ), and a pollen load comparable to that of bees (Table 1). The greater pollen load in O. jucunda is likely due to their denser body hair compared to the other beetles, a pattern consistent with findings in bees and flies (Cullen et al. 2021 ). Additionally, O. jucunda exhibits superior flight ability, which may enhance its foraging range and contribute to the diversity of pollen species it carries. Notably, Citrus pollen from a nearby orchard about 1 km from the study site prominently adhered to their bodies (Fig. S1f). On the other hand, the 'other beetles'—including 13 species spanning a diverse range of body forms, from slender, long-bodied beetles with elongated legs to compact, robust beetles with sturdy shells— carried a pollen load similar to that of hoverflies (Table 1) and exhibited the second-lowest pollen species diversity (Fig. 3 ), suggesting that they may be more efficient pollinators than O. jucunda in terms of reducing the risk of HPT. Thus, O. jucunda should be considered a separate functional group from the 'other beetles,' characterized by denser body hair, a greater capacity to carry pollen over long distances, but a higher potential for HPT. We observed striking differences in pollen species composition and diversity between the two lepidopteran groups, i.e., butterflies and nocturnal moths, while their proportions of Ligustrum pollen remained similar (Figs. 1 – 3 ). Butterflies exhibited a high diversity of body pollen species (Fig. 3 ), likely reflecting their strong flight ability (Herrera 1987 ). Supporting this, the Asteraceae species whose pollen was abundant on butterfly bodies (Fig. S1j) did not match any of the 11 Asteraceae species recorded within a 1 km radius of our site (Table S2). Previous studies have also found that butterflies carry the most diverse pollen loads among pollinators (Alarcón 2010 ; Zhao et al. 2019 ). In contrast, nocturnal moths carried significantly fewer pollen species than other pollinators (Fig. 3 ). However, their body pollen was primarily composed of L. obtusifolium and Citrus sp. (Fig. S1k), the latter of which was located at least 250 m from our site (Table S2). This suggests that low pollen species diversity in moths may not necessarily indicate limited flight ability. Instead, it may reflect a scarcity of nectar-producing flowers at night or high flower constancy in nocturnal moths driven by their odor-guided foraging (Brantjes 1978 ). These differences imply that butterflies and nocturnal moths play distinct roles as pollinators, particularly in their potential to induce HPT. Supporting this, Teng et al. ( 2024 ) reported that nocturnal moths, compared to diurnal pollinators, tend to interact with fewer flower species, often leading to greater pollen germination. While previous studies on nocturnal moth pollination have primarily focused on hawkmoths (Sphingidae), our data on settling moths reinforce the growing recognition that these non-hovering nocturnal pollinators also play an important role (Hahn and Brühl 2016 ; Walton et al. 2020 ; Singh et al. 2022 ). It is important to note that the extent of pollen heterospecificity observed in each pollinator group can vary across space and time. Network studies have often revealed significant variation in species interactions and module structures, which are influenced by climate conditions, plant diversity, pollinator abundance, and the morphological traits of both flowers and pollinators (Alarcón et al. 2008 ; Watts et al. 2016 and references therein). These factors should not be overlooked in future studies, although Cullen et al. ( 2021 ) found that insect morphological and ecological traits have a greater impact on insect body pollen than spatial and temporal variation in floral resource availability. Furthermore, pollen species composition and diversity on pollinator bodies provide only a partial perspective on the actual HPT risk, and should be complemented by behavioral observations such as visitation frequency and movement between plant species. Another key consideration is that this study evaluates pollinators solely from the perspective of HPT. To fully understand their impact on pollen transport efficiency, additional factors must be considered. For example, while bumble bees may have a low tendency to cause HPT, they often waste a significant portion of the pollen they collect by harvesting or grooming it off (Harder and Thomson 1989 ; Schlindwein et al. 2005 ; Weinman et al. 2023 ), and they tend to stay longer on individual plants than other pollinators, which can lead to increased geitonogamous self-pollination (Travis and Kohn 2023 ). Moreover, for some plant species, the negative effects of increased HPT may be outweighed by the benefits of enhanced conspecific pollen transfer. Even pollinators carrying pollen from multiple species may still provide a net benefit to plants blooming in harsh environments (Gavini et al. 2021 ; Lopes et al. 2021). We should also note that groups with comparable levels of pollen species diversity, such as nocturnal moths and worker bumble bees, often exhibited marked differences in the combination of richness and evenness (Fig. 3 ). The consequences of such variation in heterospecific pollen composition for plant reproductive success may depend not only on the total diversity of pollen grains but also on the phylogenetic relatedness among pollen donors (Arceo-Gómez and Ashman 2016 ; Streher et al. 2020 ). With these caveats in mind regarding how different body pollen profiles affect plant reproduction, our findings offer several intriguing implications. The most striking is that when pollinator quality is reassessed based on the likelihood of causing HPT, a single conventionally recognized functional group may actually be divided into multiple groups. If this is the case, different phenotypes could evolve within a group of flowers classified as a 'bee-pollinated,' 'beetle-pollinated,' or 'fly-pollinated' system. For example, flowers frequently visited by pollinators with higher HPT propensity may be more likely to evolve floral traits that reduce the risk of HPT with closely related species blooming nearby—such as unique floral colors or scents that promote individual specialization in foraging (Gumbert et al. 1999 ) or traits like prior selfing (Randle et al. 2018 ; Katsuhara and Ushimaru 2019 ). In other cases, heterospecific pollen deposition may reduce opportunities for effective pollination by more faithful visitors, driving the evolution of filter traits that actively exclude low-fidelity pollinators (Ohashi et al. 2021 ). For example, a 'bee-pollinated' flower may evolve traits that exclude solitary bees to minimize HPT while still allowing visits from bumble bees. In such cases, grouping all bees into a single functional group could obscure important evolutionary consequences, particularly those related to the evolution of floral filters. In sum, we demonstrated significant variation in heterospecificity of body pollen both among and within conventional functional groups of pollinators through an exhaustive comparison of pollen loads on individual insect bodies. Our findings highlight the need to place greater emphasis on the behavioral aspects of pollinators, alongside morphological traits, when defining their functional groups. Building on our current findings, a better understanding of how such diversity in pollen heterospecificity affects plant reproduction could contribute to a more comprehensive view of pollinator quality, their ecological significance, and, in turn, their evolutionary relationships with floral phenotypes. Declarations - The authors have no relevant financial or non-financial interests to disclose. - The authors have no competing interests to declare that are relevant to the content of this article. - All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. - The authors have no financial or proprietary interests in any material discussed in this article. Ethical approval No formal ethical approval was required for this research, as it involved the study of insects, which are not subject to formal ethics approval processes in Japan. Data Availability The data are available on Figshare: https://figshare.com/s/83950d8b1bece4dbfd12 (Terada & Ohashi, 2025). Note: this is a private link; a DOI will be provided upon acceptance. Acknowledgments We thank Yukie Sato and Mitsuru Hirota for their valuable advice and discussions. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7135148","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515618272,"identity":"c4882c0f-6ba9-4fea-9631-2ad1fafe7f99","order_by":0,"name":"Kohei Terada","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Kohei","middleName":"","lastName":"Terada","suffix":""},{"id":515618273,"identity":"d7789dec-47c8-488d-a3e1-c715ea30c993","order_by":1,"name":"Kazuharu Ohashi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIie3QsUoDMRjA8S8c1CXF9SuH11e4I1ALlT5LwkG79HyCchSEuJzOVyo+Qzd1yxGwS3HWzSeQc5HeUpqqiJVG6iYl/yl88ONLAuBy/cMQKAAZYRB6QNTXZB0Z/U6YIaCA705ATOEnsdU4vyheq9t2/+bAU4osUjHOk6KEYRe8yfY1Pn2I/fock7uzGjdbtJjgaYxwHwO5UltJgIPQJxKTqaahIUpcmwlCTQHJuY2wqpLYDz9IuiZsAUs78XHQwrpE/kk8czEzIdJOGtm81TEken8L72k2zl56bXEZU9tbcJaxp0qmzeNDXTyXJ+lRPkv0Y/nWDSLLj23Gvx1olO8gNmvin4nL5XLtZytP510UcBFz0gAAAABJRU5ErkJggg==","orcid":"","institution":"University of Tsukuba","correspondingAuthor":true,"prefix":"","firstName":"Kazuharu","middleName":"","lastName":"Ohashi","suffix":""}],"badges":[],"createdAt":"2025-07-16 03:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7135148/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7135148/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91663327,"identity":"31338952-58f1-4d4e-a2a5-208cef5be456","added_by":"auto","created_at":"2025-09-18 22:57:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":158879,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of heterospecific (non-\u003cem\u003eLigustrum\u003c/em\u003e) pollen on the body of each of the 11 insect groups. Data from 2020. Values represent the back-transformed proportions of heterospecific pollen per insect. Marginal (model-adjusted) means ± 95% CI are shown. The difference in means was tested for each pair of groups. Means sharing a letter are not significantly different at the 0.05 level after adjustment by the Benjamini-Hochberg method.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7135148/v1/4f4ad355931def98f59e9d01.png"},{"id":91663323,"identity":"888e6d39-a6c3-429e-9570-751fccf08197","added_by":"auto","created_at":"2025-09-18 22:57:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":331890,"visible":true,"origin":"","legend":"\u003cp\u003eNMDS ordination plot comparing body pollen assemblages (species level) of the 11 insect groups visiting \u003cem\u003eL. obtusifolium\u003c/em\u003e, based on Bray-Curtis dissimilarity. Convex hull polygons delineate the 11 insect groups, and symbols represent individuals within each group. PERMANOVA revealed a significant difference among the 11 groups (P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7135148/v1/8ed89acf1adb468a5bf3fd7d.png"},{"id":91663424,"identity":"0fd96d21-bce4-4d7d-8977-e892c49595ab","added_by":"auto","created_at":"2025-09-18 23:05:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":348860,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between the richness and evenness of body pollen species across 11 insect groups. Richness and evenness were evaluated using pollen species number and Sheldon’s equitability index, respectively. Symbols and error bars represent means and standard errors (SEs) for each group. Numbers indicate the average Shannon-Wiener index (H'), calculated as log(richness × evenness), for individual groups. Dashed lines represent isoclines showing combinations of richness and evenness that yield the same Shannon-Wiener index (H') as observed for the respective groups.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7135148/v1/b5b37d1f901cf83e49cb200d.png"},{"id":91663574,"identity":"2563400d-ab97-465a-91c1-91553ee54477","added_by":"auto","created_at":"2025-09-18 23:13:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1264572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7135148/v1/1b0095ea-7b09-4419-8c2b-78ff8240747b.pdf"},{"id":91663326,"identity":"d8d3ca4e-fff7-4736-bb28-8ba5f3aecf4b","added_by":"auto","created_at":"2025-09-18 22:57:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":25950,"visible":true,"origin":"","legend":"","description":"","filename":"Table.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7135148/v1/6e3d489e173d97ec1dfd4d1e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Heterospecificity of body pollen varies across and within functional groups of pollinators","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSelection pressures on floral phenotypes exerted by distinct types of pollinators are widely regarded as key drivers of floral diversification and convergence. To capture presumed similarities in these selective forces, pollinators have traditionally been grouped into functional groups (Vogel \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1954\u003c/span\u003e; Faegri and van der Pijl 1979; Fenster et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), which often corresponded to taxonomic classifications such as bees, flies, butterflies, moths, beetles, birds, and bats (Fenster et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This taxonomically oriented framework likely stems from the assumption that a pollinator's contribution to plant reproduction is primarily determined by the amount of pollen it removes from anthers and deposits onto stigmas of other plants (Bierzychudek 1981; Ne'eman et al. 2010; Thomson et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Johnson and Harder \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and that animals with similar sizes and morphologies\u0026mdash;often (though not always) shared within taxonomic groups\u0026mdash;therefore exhibit comparable levels of pollen transport and, by extension, exert similar selective pressures on flowers (Fenster et al \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile this approach has some practical utility, it may overlook important differences in the \u003cem\u003equality\u003c/em\u003e of pollen transfer among pollinators, which are strongly influenced by their foraging behavior among flowers and plants. For example, studies have shown that pollinators can differ markedly in the proportion of self- versus outcross pollen on their bodies (Hasegawa et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and in the genetic diversity of pollen donors they transfer (Valverde et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition to these genetic components, accumulating evidence suggests that a pollinator\u0026rsquo;s contribution also depends on how frequently it moves between plant species, as this increases the likelihood of heterospecific pollen transfer (HPT)\u0026mdash;a process that can reduce male fitness by diverting pollen to heterospecific stigmas (Muchhala and Thomson \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and female fitness through mechanisms such as stigma clogging (Galen and Gregory \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), stigma closure (Waser and Fugate \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), allelopathic or physical interference with pollen germination and growth (Buchman and Campbell \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Murphy \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), or ovule usurpation and hybridization (Harder et al. 1993; Burgess et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe propensity to induce HPT can vary among pollinators, depending on various factors, including their perceptual and cognitive abilities (Latty and Trueblood \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), dietary breadth (Brosi \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), flower constancy (Goulson and Wright \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Chittka et al. 1999; Amaya-M\u0026aacute;rquez 2009), foraging range (Gr\u0026uuml;ter and Hayes \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), sampling frequency (Heinrich \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Dunlap et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), pollen foraging behavior (Russell et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), among others. Some suggestive observations have been made previously in this regard. For example, butterflies have been observed flying longer distances than other flower visitors, often bypassing nearby plants (Schmitt \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Herrera \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), potentially increasing the likelihood of cross-species pollen transfer (Alarc\u0026oacute;n \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A study on 85 bee taxa found that eusocial bees exhibit 67.5% greater pollen diet diversity compared to noneusocial bees (Devkota et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), indicating that they are inferior pollinators in terms of the heterospecificity of body pollen. Moreover, varying tendencies toward HPT may exist even within conventionally recognized functional groups of pollinators. For example, male bumble bees have been observed to collect pollen from a narrower range of flowers than workers (Wolf and Moritz \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite these indications, our current knowledge is still limited and fragmented regarding how the propensity to cause HPT varies among and within different groups of pollinators.\u003c/p\u003e\u003cp\u003eIn order to gain further insight into this issue, we conducted a field survey in a Japanese \u003cem\u003esatoyama\u003c/em\u003e landscape to quantify and compare the composition of body pollen carried by a diverse range of insects visiting flowers of border privet, \u003cem\u003eLigustrum obtusifolium\u003c/em\u003e Siebold et Zucc. (Oleaceae), and to assess their potential to induce HPT. \u003cem\u003eLigustrum obtusifolium\u003c/em\u003e is a generalist flowering species that attracts a broad range of insect pollinators. We therefore assumed that visitors collected from a few nearby \u003cem\u003eL. obtusifolium\u003c/em\u003e plants would share overlapping foraging ranges and, to some extent, access to a common pool of co-flowering species. This assumption allowed us to quantitatively compare their inherent propensity to forage across multiple species\u0026mdash;a comparison that would have been less straightforward if insects had been sampled from different plant species or separate locations. We examined the composition of pollen species found on each pollinator's body, and compared (1) the proportion of heterospecific (non- \u003cem\u003eLigustrum\u003c/em\u003e) pollen, (2) the composition of pollen species, and (3) the diversity of pollen species. Drawing from these data, we discuss how the observed variations in body pollen heterospecificity, or the propensity to induce HPT, could offer new insights into conventional views on functional groups of pollinators, which are largely based on taxonomy or morphological/size similarities.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eInsect sampling\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eLigustrum obtusifolium\u003c/em\u003e is a small deciduous shrub or tree that can reach a height of 3\u0026ndash;6 m. In early summer, it produces hermaphroditic flowers that are arranged in dense inflorescences. Each flower has a tubular corolla (10 mm in length, 5 mm in diameter) with four lobes, typically white or cream-colored. Its nectar and a sweet odor strongly attract insect visitors of various taxa, including bees, wasps, flies, beetles, butterflies, and nocturnal moths (Yokoi et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ikenoue and Kanai, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Our preliminary survey at this site in 2019 identified 125 insect species from six orders, 37 families, and 109 genera (Table S1). This pollinator-generalist flower often serves as the hub species within local pollination networks, making it suitable for the purpose of this study.\u003c/p\u003e\u003cp\u003eWe conducted the study in \u003cem\u003eSusomi no Mori\u003c/em\u003e (meaning 'foothill forest'), a small secondary forest at the foot of Mt. Tsukuba, Ibaraki, Japan (36\u0026deg;11'34.1\" N, 140\u0026deg;06'44.6\" E), surrounded by traditional farmland, including rice paddies. This forest has been managed by the NPO Tsukuba Environmental Forum since 2006 to conserve its \u003cem\u003esatoyama\u003c/em\u003e landscape and sustain local cultural heritage. From 24 to 31 May 2020, we conducted around-the-clock sampling of flower visitors on three closely growing \u003cem\u003eL. obtusifolium\u003c/em\u003e plants. We observed these trees during the day (for a total of 30 hours) and the night (for a total of 8 hours), capturing all the flower visitors using an insect net. At least 25 individuals were collected for each of the 11 insect groups described in the following. Nocturnal observations were made using a headlamp of \u0026gt;\u0026thinsp;350 lumens (LEDLenser\u0026reg; H14R, Solingen, Germany) covered with red cellophane. We placed the captured insects separately in centrifuge tubes of appropriate sizes, ranging from 1.5 ml to 30 ml, or triangular paper envelopes for lepidopterans. We then transported them back to the laboratory in a cooler box and preserved them in a freezer set to -20\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe sampled insects were classified into 11 groups based on their distinct behavior and morphology: worker bumble bees (\u003cem\u003eBombus ardens\u003c/em\u003e), male bumble bees (\u003cem\u003eB. ardens\u003c/em\u003e), mining bees (\u003cem\u003eAndrena\u003c/em\u003e spp.), the other bees, wasps, hairy scarab beetles (\u003cem\u003eOxycetonia jucunda\u003c/em\u003e), the other beetles, hoverflies (Syrphidae), the other flies, butterflies, and nocturnal moths. Bumble bees are grouped separately from other bees due to their high cognitive abilities, which are associated with efficient tactics for locating and obtaining resources, such as individual specialization (Heinrich \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1976a\u003c/span\u003e) and memory-based spatial foraging (Cartar \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Ohashi and Thomson \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Ogilvie and Thomson \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Taking advantage of their highly frequent visits, we further separated them into males and workers (females) to see if there were differences derived from their foraging requirements (Wolf and Moritz \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Among other bees, we also treated mining bees (\u003cem\u003eAndrena\u003c/em\u003e spp.) as a distinct group because this genus comprises numerous oligolectic species (Danforth \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). One abundant scarab species, \u003cem\u003eO. jucunda\u003c/em\u003e, was distinguished from other coleopterans based on its distinctly dense hair and strong flight capabilities. For dipterans, we separated hoverflies from other (muscoid and oestroid) flies because of their strong dependence on floral resources and their selective foraging (Larson et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Inouye et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Finally, butterflies and nocturnal moths were distinguished based on their activity time, reliance on different sensory modalities (i.e., vision and olfaction) (Brantjes \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Cunningham et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Balkenius et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and the resulting expectations regarding their foraging repertoires. Note that nocturnal moths on \u003cem\u003eL. obtusifolium\u003c/em\u003e were all settling moths, which land on flowers to probe nectar. No hawkmoths (Sphingidae) were observed visiting flowers of \u003cem\u003eL. obtusifolium\u003c/em\u003e. Diurnal moths were excluded from the analysis due to the small sample size.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePollen identification and counting\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePollen grains were collected from insect bodies following the method described by Nikkeshi et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), i.e., by rinsing the insect body with a sucrose solution. We carefully avoided any contamination with pollen that could not be used for pollination, by excluding samples from insects that had regurgitated or excreted pollen, as well as those from female bees with visibly distinct sizes of pollen loads on their corbiculae (less than 10% of the collected individuals). Each insect was placed in an appropriate size centrifuge tube (1.5\u0026ndash;3.0 ml) with a 0.4 mol/l sucrose solution. After shaking the tube up and down by hand, being careful not to destroy the insect body, we agitated the tube for 1 minute on a vortex mixer (Tube Mixer Trio HM-1F, AS ONE). Next, we sonicated the tube for 5 minutes in an ultrasonic bath (UT-106H, SHARP). Finally, we agitated the tube again on a vortex for 1 minute, after which we shook the tube once by hand. This method has been shown to separate 96.5% or more of the total pollen attached to the body surface of an insect (Nikkeshi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For each insect, we prepared five microscope slides, each of which was made from 10 \u0026micro;l of pollen suspension. We then examined the slides under a microscope (OLYMPUS CX43) equipped with a microscope camera (AdvanVision AdvanCam-HD2s) to identify and count pollen grains.\u003c/p\u003e\u003cp\u003ePollen identification was performed by referring to a collection of pollen slides and microscopic images of flowering species around our study site. Before and during the flowering season of \u003cem\u003eL. obtusifolium\u003c/em\u003e (15\u0026ndash;31 May 2020), we conducted an exhaustive search by foot for co-flowering species within a 1-km radius of the \u003cem\u003eL. obtusifolium\u003c/em\u003e trees. Each flowering species was identified, and the coordinates of the individual closest to our focal \u003cem\u003eL. obtusifolium\u003c/em\u003e trees were recorded using a handheld GPS receiver (model: eTrex 30x; Garmin, Olathe, KS, USA) with an accuracy of less than two meters (Table S2). The anthers were collected from these flowers just before/after anthesis and placed in microtubes (1.5 ml) with forceps. In the laboratory, we stored the anthers in a dried condition by opening the lids of microtubes and placing them in a sealed container with calcium chloride. Similarly to the collection of insect body pollen described above, the pollen in these anthers was dissolved in 1 ml of a 0.4 mol/l sucrose solution by agitating for 1 minute on a vortex mixer, followed by 5 minutes of sonication in an ultrasonic bath. We prepared a microscopic slide from 10 \u0026micro;l of each pollen suspension and captured images using the same microscope camera system.\u003c/p\u003e\u003cp\u003eAfter counting the pollen on five slides for each insect, the total number of pollen grains of the i-th plant species, NP\u003csub\u003ei\u003c/sub\u003e, was estimated as follows:\u003c/p\u003e\u003cp\u003eNP\u003csub\u003ei\u003c/sub\u003e = (No. of pollen grains of the i-th species in a 50-\u0026micro;l suspension) x (Total volume of the pollen suspension in microliter unit) / 50 (1).\u003c/p\u003e\u003cp\u003eUsing the above method, Nikkeshi et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have shown that 95% or more of the pollen species on the body surface of a single insect can be detected.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eTo contrast and compare the potential impact of each pollinator group on HPT, we focused on three aspects of heterospecificity of insect body pollen, i.e., i) proportion of heterospecific pollen (=\u0026thinsp;pollen other than \u003cem\u003eL. obtusifolium\u003c/em\u003e), ii) pollen species composition, and iii) pollen species diversity, as described in more detail below. All analyses were performed using R (R Core Team \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and all graphs were created using the ggplot2 package (Wickham \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFirst, we determined whether the proportion of heterospecific pollen differed among pollinator groups visiting \u003cem\u003eL. obtusifolilum\u003c/em\u003e by fitting a generalized linear-mixed model (GLMM) with a logit link function and a binomial error distribution. The proportion of heterospecific pollen, calculated as the number of heterospecific pollen grains other than \u003cem\u003eL. obtusifolium\u003c/em\u003e devided by the total number of pollen grains on a insect's body (\u0026sum;NP\u003csub\u003ei\u003c/sub\u003e), was used as the response variable. We considered pollinator group as a fixed effect and individual insects as a random effect. For the model fitting, the calculation of marginal and conditional R\u003csup\u003e2\u003c/sup\u003e (Nakagawa and Schielzeth \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), the type-II Wald chi-square tests, and the estimation of marginal (model-adjusted) means and 95% confidence intervals with the comparison of means, we used \u003cem\u003eglmer\u003c/em\u003e in \u0026lsquo;lme4\u0026rsquo; (Bates et al. 2014), \u003cem\u003er.squaredGLMM\u003c/em\u003e in \u0026lsquo;MuMIn\u0026rsquo; (Barton and Barton \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eAnova\u003c/em\u003e in \u0026lsquo;car\u0026rsquo; (Fox et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and \u003cem\u003eemmeans\u003c/em\u003e with \u003cem\u003epairs\u003c/em\u003e in \u0026lsquo;emmeans\u0026rsquo; (Searle et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Lenth et al. 2020), respectively.\u003c/p\u003e\u003cp\u003eSecond, we investigated whether and to what extent the species composition of body pollen varied among different pollinator groups. To obtain a dissimilarity matrix reflecting the compositional dissimilarity among pollinator groups, we applied the Bray-Curtis distance to the pollen species matrix using the \u003cem\u003evegdist\u003c/em\u003e in \u0026lsquo;vegan\u0026rsquo; (Oksanen et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Next, we performed a non-metric multidimensional scaling (NMDS, Kruskal \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1964\u003c/span\u003e) using \u003cem\u003emetaMDS\u003c/em\u003e function in \u0026lsquo;vegan\u0026rsquo; to reduce the dimensionality of species composition to two while minimizing the departure from the distance relationships in the original data. Finally, we performed a PERMANOVA to test whether pollen species composition differed among pollinator groups using \u003cem\u003eadonis\u003c/em\u003e function in \u0026lsquo;vegan\u0026rsquo;.\u003c/p\u003e\u003cp\u003eFinally, we assessed the diversity of pollen species on each insect's body, as insects with a more diverse body pollen may deposit more foreign pollen onto the stigmas. The diversity of body pollen species was evaluated with Shannon-Wiener Index (Shannon \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1948\u003c/span\u003e). Biodiversity generally reflects the two often independent components, i.e., richness (number of species) and evenness (equitability of the proportional abundances of those species) (Sheldon \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Hurlbert \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Soininen et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tuomisto \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, we compared the characteristics of the observed species diversity of body pollen among 11 pollinator groups by plotting the data on a two-dimensional space with two orthogonal axes representing richness and evenness. We measured evenness using the Sheldon's equitability index (Sheldon \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1969\u003c/span\u003e) calculated as:\u003c/p\u003e\u003cp\u003eE\u0026thinsp;=\u0026thinsp;\u003cem\u003ee\u003c/em\u003e\u003csup\u003eH'\u003c/sup\u003e/S (2),\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003ee\u003c/em\u003e is the base of the natural logarithms, H' is the Shannon-Wiener index, and S is pollen species richness on the insect's body. The quantity \u003cem\u003ee\u003c/em\u003e\u003csup\u003eH'\u003c/sup\u003e is the minimum number of equally common species that could yield the observed diversity H'. To correct for differences in the amount of body pollen among insects, we computed \u003cem\u003ee\u003c/em\u003e\u003csup\u003eH'\u003c/sup\u003e and S based on rarefaction for a sample size of 10 pollen grains, using \u003cem\u003eiNEXT\u003c/em\u003e function in \u0026lsquo;iNEXT\u0026rsquo; (Hsieh et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eWe examined 331 insects and recorded between 10 and 1,122,500 pollen grains on their body surfaces. The pollen grains found on an insect\u0026rsquo;s body contained up to eight plant species. The number of body pollen grains per individual in each insect group is summarized in Table\u0026nbsp;1. The species profile of body pollen across 11 insect groups is shown in Fig. S1. The following sections present analyses focusing on three aspects of body pollen heterospecificity.\u003c/p\u003e\u003cp\u003e\u003cem\u003eProportion of heterospecific pollen\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe observed a significant variation in the proportion of heterospecific (non-\u003cem\u003eLigustrum\u003c/em\u003e) pollen among the 11 observed insect groups (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;146.5, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Type II Wald chi-square test; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The variation in the proportion of heterospecific pollen among individuals was explained by the insect group, accounting for 29% of the variation, while the majority of the remaining variation was attributed to a random effect, namely, insect individuals (marginal R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.29, 0.99\u0026thinsp;\u0026lt;\u0026thinsp;conditional R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;1; Nakagawa \u0026amp; Schielzeth\u0026rsquo;s pseudo-R\u003csup\u003e2\u003c/sup\u003e for GLMM).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe proportion of heterospecific pollen was particularly high in mining bees and hairy scarab beetles. In contrast, bumble bees exhibited much lower heterospecificity, with workers showing significantly higher values than males (Benjamini-Hochberg adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). We also observed significant variation in heterospecific pollen proportion within the same order or superfamily. Specifically, mining bees, consisting of three Andrena species (Andrenidae), had a significantly higher proportion of heterospecific pollen than bumble bees (\u003cem\u003eB. ardens\u003c/em\u003e) and the 'other bees' group (13 species from Apidae, Megachilidae, Halictidae, and Colletidae) (Benjamini-Hochberg adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Interestingly, male bumble bees showed significantly lower heterospecificity than workers (male: N\u0026thinsp;=\u0026thinsp;30, marginal mean\u0026thinsp;\u0026plusmn;\u0026thinsp;95% CI\u0026thinsp;=\u0026thinsp;0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06; worker: N\u0026thinsp;=\u0026thinsp;30, 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19). Hairy scarab beetles (\u003cem\u003eO. jucunda\u003c/em\u003e) exhibited a significantly higher proportion of heterospecific pollen than the 'other beetles' group (13 species from Scarabaeidae, Cerambycidae, Elateridae, Cantharidae, Nitidulidae, Melyridae, Oedemeridae, and Carabidae) (Benjamini-Hochberg adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Although male hairy scarab beetles showed slightly lower heterospecificity than females (male: N\u0026thinsp;=\u0026thinsp;13, marginal mean\u0026thinsp;\u0026plusmn;\u0026thinsp;95% CI\u0026thinsp;=\u0026thinsp;0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29; female: N\u0026thinsp;=\u0026thinsp;17, 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14), this trend was not statistically significant, likely due to small sample sizes (P\u0026thinsp;\u0026gt;\u0026thinsp;0.17, Type II Wald chi-square test).\u003c/p\u003e\u003cp\u003eOn the other hand, the difference in heterospecificity of body pollen was not significant between the two Diptera groups, i.e., hoverflies (nine species from Syrphidae) and the 'other flies' (8 species from Muscidae, Calliphoridae, Anthomyiidae, and Conopidae) (Benjamini-Hochberg adjusted P\u0026thinsp;=\u0026thinsp;0.070). Likewise, we found no significant difference between butterflies (nine species from Papilionidae, Pieridae, Nymphalidae, and Hesperiidae), and nocturnal moths (17 species from Crambidae, Geometridae, and Noctuidae) (Benjamini-Hochberg adjusted P\u0026thinsp;=\u0026thinsp;0.26).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePollen species composition\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePollen species composition varied significantly among the 11 observed insect groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, PERMANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This partially reflects the pollen compositional similarities within each insect group. Of particular note, both worker and male bumble bees exhibited highly converged profiles of pollen species among individuals, as demonstrated by the narrow polygonal areas shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The level of similarity among individuals in these groups was more pronounced than in hairy scarab beetles, which also consisted of a single insect species. Close similarity within the same group was not always observed, however. Hoverflies and the 'other flies' showed noticeable within-group variations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Because each group includes nine and eight species, respectively, a significant portion of this variability is likely attributed to interspecific differences. Similar trends were also found in the two non-\u003cem\u003eBombus\u003c/em\u003e bee groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIt should be noted that considerable differences in pollen species composition were observed even among closely related insect groups. The compositional difference between the two Diptera groups, i.e., hoverflies and the 'other flies', as well as between the two bee groups, i.e., mining bees and the 'other bees', falls into this category (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Yet another example is the two groups of Lepidoptera, i.e., butterflies and nocturnal moths (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This difference reflects that, except for \u003cem\u003eLigustrum\u003c/em\u003e pollen, butterflies often carried a large amount of pollen from a single species of Asteraceae (64% of heterospecific pollen), while nocturnal moths often carried a large amount of pollen from a single species of \u003cem\u003eCitrus\u003c/em\u003e (91% of heterospecific pollen) (see Fig. S1j, k for more details).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePollen species diversity\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the relationship between the richness and evenness of body pollen species across 11 insect groups. Consistent with the other two analyses, male bumble bees exhibited the lowest species diversity of body pollen (H' = 0.33). Worker bumble bees were not the second lowest in terms of pollen species diversity (H' = 0.63) because their relatively high richness counteracted the low evenness caused by their highly biased pollen species composition. In contrast to bumble bees, the other two bee groups, i.e., mining bees and the 'other bees', both exhibited the highest levels of pollen species diversity (mining bees: H' = 0.94, other bees: H' = 0.90). All four groups of bees carried several pollen species on their bodies, but in bumble bees, species abundance was highly skewed toward one or two species, whereas in non-\u003cem\u003eBombus\u003c/em\u003e bees, pollen species were more evenly distributed (see also Fig. S1a-d). For another hymenopteran group, wasps, pollen species diversity was intermediate (H' = 0.73). Among the hymenopterans, this group had the lowest pollen richness but the highest evenness.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePatterns of pollen species diversity varied within the other insect orders as well. Among beetles, hairy scarab beetles exhibited higher diversity than the 'other beetles' (hairy scarab beetles: H' = 0.74, 'other beetles': H' = 0.55), primarily due to differences in species richness (see also Fig. S1f, g). A similar contrast was found within Lepidoptera, where butterflies exhibited one of the highest levels of pollen species diversity (H' = 0.93) among all insect groups, whereas nocturnal moths showed lower diversity (H' = 0.64), at a similar level to that of worker bumble bees.\u003c/p\u003e\u003cp\u003eIn contrast to these patterns, we observed little variation in pollen species diversity between the two dipteran groups, with hoverflies (H' = 0.77) and 'other flies' (H' = 0.80) exhibiting similar, intermediate levels. This uniformity in diversity sets Diptera apart from the other insect orders analyzed, despite variations in their pollen-carrying capacities (Table\u0026nbsp;1) and pollen species composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study revealed that the 11 insect groups visiting \u003cem\u003eL. obtusifolium\u003c/em\u003e flowers differ significantly in all three aspects: the proportion of heterospecific pollen, pollen species composition, and pollen species diversity. Substantial differences were observed not only among phylogenetically distant taxa but also among closely related taxa and even between sexes of the same species. These findings support our initial hypothesis that groupings of pollinators based primarily on taxonomy or morphological and size similarities may not adequately reflect their pollination quality in terms of HPT potential\u0026mdash;a factor that may have largely been overlooked in the literature\u0026mdash;and reinforce the importance of considering the behavioral aspects of pollinators when defining their functional groups (Maubecin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Below, we discuss the major trends in the three-dimensional heterospecificity of body pollen, both among and within conventional functional groups, as well as their possible consequences for plant reproduction.\u003c/p\u003e\u003cp\u003eThe body pollen of bumble bees exhibited low species diversity and little individual variation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), being predominantly composed of \u003cem\u003eL. obtusifolium\u003c/em\u003e and a few other species (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1a, b). This pattern was particularly pronounced in males, whose pollen species composition was far more uniform than that of workers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Such extreme pollen constancy suggests that bumble bees not only serve as one of the insect groups carrying the largest amounts of pollen (Table\u0026nbsp;1) but also contribute little to heterospecific pollen transfer (HPT), both for \u003cem\u003eL. obtusifolium\u003c/em\u003e and for co-flowering plant species.\u003c/p\u003e\u003cp\u003eThis finding may be explained by individual bumble bees' tendency to develop foraging preferences for one or a few species (Heinrich \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1976a\u003c/span\u003e), their site fidelity to small areas (Thomson \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Cartar \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Ogilvie and Thomson \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), or their flower constancy\u0026mdash;where they make consecutive visits to the same flower species while disregarding others, especially when that species forms large clusters (Chittka et al. 1999; Ishii \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Takagi and Ohashi \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In particular, the significantly lower pollen species diversity in males compared to workers (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) may correspond to their spatial foraging patterns. Ogilvie and Thomson (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) observed that male bumble bees tend to forage within smaller, more localized areas than workers, which could contribute to their extreme pollen uniformity. This explanation seems plausible, considering that our \u003cem\u003eL. obtusifolium\u003c/em\u003e trees produced enough flowers to saturate individual foragers.\u003c/p\u003e\u003cp\u003eIn contrast, the body pollen of mining bees (\u003cem\u003eAndrena\u003c/em\u003e spp.), which had the highest per-insect pollen load (Table\u0026nbsp;1), exhibited the greatest species diversity among the 11 insect groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and was often dominated by non-\u003cem\u003eLigustrum\u003c/em\u003e species (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1c). This finding contradicts the common perception of the family Andrenidae as comprising numerous oligolectic species (Danforth \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), as well as the recognized pattern that solitary bees exhibit lower pollen diet diversity than eusocial bees (Devkota et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, it may suggest that all the species found on \u003cem\u003eL. obtusifolium\u003c/em\u003e were polylectic\u0026mdash;a trait that have evolved later in this lineage (Larkin et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Similar trends in body pollen were observed for the 'other bees' group, which showed higher species diversity than bumble bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), although \u003cem\u003eLigustrum\u003c/em\u003e pollen accounted for more than 50% of the total (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNote that both of these bee groups also exhibited greater individual variation in pollen species composition than bumble bees (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), although the current data do not rule out the possibility that this variation is largely due to interspecific or interfamily differences. The low individual variation observed in bumble bees, despite their generalist foraging nature (Watts et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Russell et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Peat et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), may be partly due to the abundant floral resources of \u003cem\u003eL. obtusifolium\u003c/em\u003e, which could have reduced the need for resource partitioning among individuals (Heinrich \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1976b\u003c/span\u003e). However, since the other bee pollinators were exposed to the same floral environment, it is likely that these solitary bee species inherently exhibit greater individual variation than bumble bees.\u003c/p\u003e\u003cp\u003eCompared to bees, wasps carried fewer pollen species on their bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and tended to transport less pollen overall (Table\u0026nbsp;1). Their pollen-carrying characteristics more closely resembled those of the 'other flies' group rather than the three bee groups. This reduced pollen load may be, in part, due to their lower body hair density and their greater reliance on carnivory over floral resource use. Additionally, the species composition of wasp body pollen was distinct from that of all other insect groups, often being dominated by arrowwood (\u003cem\u003eViburnum dilatatum\u003c/em\u003e) (Fig. S1e). This pattern may reflect wasps' preference for dish-shaped flowers with exposed nectaries, such as those of arrowwood, due to their relatively short mouthparts (F\u0026aelig;gri and van der Pijl, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1979\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast to this cross-taxon similarity, the two dipteran groups\u0026mdash;hoverflies and the 'other flies'\u0026mdash;exhibited distinct body pollen profiles in both total pollen amount (Table\u0026nbsp;1) and species composition (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and S1h, i). Notably, hoverflies carried five times more pollen than the other flies (Table\u0026nbsp;1). These differences may reflect a higher floral dependence and visitation frequency in hoverflies compared to other flies. However, these differences do not necessarily indicate that one group is more likely to cause HPT than the other, as they were similar both in the proportion of \u003cem\u003eLigustrum\u003c/em\u003e pollen and pollen species diversity (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Hoverflies may be considered a distinct functional group\u0026mdash;separate from other flies\u0026mdash;and may provide more effective pollination without increasing the risk of HPT, particularly in plant species where HPT is not a major concern.\u003c/p\u003e\u003cp\u003eHairy scarab beetles (\u003cem\u003eO. jucunda\u003c/em\u003e) stood out from the 'other beetles' group with a lower proportion of \u003cem\u003eLigustrum\u003c/em\u003e pollen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a distinct pollen species profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), higher species diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and a pollen load comparable to that of bees (Table\u0026nbsp;1). The greater pollen load in \u003cem\u003eO. jucunda\u003c/em\u003e is likely due to their denser body hair compared to the other beetles, a pattern consistent with findings in bees and flies (Cullen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, \u003cem\u003eO. jucunda\u003c/em\u003e exhibits superior flight ability, which may enhance its foraging range and contribute to the diversity of pollen species it carries. Notably, \u003cem\u003eCitrus\u003c/em\u003e pollen from a nearby orchard about 1 km from the study site prominently adhered to their bodies (Fig. S1f). On the other hand, the 'other beetles'\u0026mdash;including 13 species spanning a diverse range of body forms, from slender, long-bodied beetles with elongated legs to compact, robust beetles with sturdy shells\u0026mdash; carried a pollen load similar to that of hoverflies (Table\u0026nbsp;1) and exhibited the second-lowest pollen species diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting that they may be more efficient pollinators than \u003cem\u003eO. jucunda\u003c/em\u003e in terms of reducing the risk of HPT. Thus, \u003cem\u003eO. jucunda\u003c/em\u003e should be considered a separate functional group from the 'other beetles,' characterized by denser body hair, a greater capacity to carry pollen over long distances, but a higher potential for HPT.\u003c/p\u003e\u003cp\u003eWe observed striking differences in pollen species composition and diversity between the two lepidopteran groups, i.e., butterflies and nocturnal moths, while their proportions of \u003cem\u003eLigustrum\u003c/em\u003e pollen remained similar (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Butterflies exhibited a high diversity of body pollen species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), likely reflecting their strong flight ability (Herrera \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Supporting this, the Asteraceae species whose pollen was abundant on butterfly bodies (Fig. S1j) did not match any of the 11 Asteraceae species recorded within a 1 km radius of our site (Table S2). Previous studies have also found that butterflies carry the most diverse pollen loads among pollinators (Alarc\u0026oacute;n \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, nocturnal moths carried significantly fewer pollen species than other pollinators (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, their body pollen was primarily composed of \u003cem\u003eL. obtusifolium\u003c/em\u003e and \u003cem\u003eCitrus\u003c/em\u003e sp. (Fig. S1k), the latter of which was located at least 250 m from our site (Table S2). This suggests that low pollen species diversity in moths may not necessarily indicate limited flight ability. Instead, it may reflect a scarcity of nectar-producing flowers at night or high flower constancy in nocturnal moths driven by their odor-guided foraging (Brantjes \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). These differences imply that butterflies and nocturnal moths play distinct roles as pollinators, particularly in their potential to induce HPT. Supporting this, Teng et al. (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that nocturnal moths, compared to diurnal pollinators, tend to interact with fewer flower species, often leading to greater pollen germination. While previous studies on nocturnal moth pollination have primarily focused on hawkmoths (Sphingidae), our data on settling moths reinforce the growing recognition that these non-hovering nocturnal pollinators also play an important role (Hahn and Br\u0026uuml;hl \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Walton et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt is important to note that the extent of pollen heterospecificity observed in each pollinator group can vary across space and time. Network studies have often revealed significant variation in species interactions and module structures, which are influenced by climate conditions, plant diversity, pollinator abundance, and the morphological traits of both flowers and pollinators (Alarc\u0026oacute;n et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Watts et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2016\u003c/span\u003e and references therein). These factors should not be overlooked in future studies, although Cullen et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that insect morphological and ecological traits have a greater impact on insect body pollen than spatial and temporal variation in floral resource availability. Furthermore, pollen species composition and diversity on pollinator bodies provide only a partial perspective on the actual HPT risk, and should be complemented by behavioral observations such as visitation frequency and movement between plant species. Another key consideration is that this study evaluates pollinators solely from the perspective of HPT. To fully understand their impact on pollen transport efficiency, additional factors must be considered. For example, while bumble bees may have a low tendency to cause HPT, they often waste a significant portion of the pollen they collect by harvesting or grooming it off (Harder and Thomson \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Schlindwein et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Weinman et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and they tend to stay longer on individual plants than other pollinators, which can lead to increased geitonogamous self-pollination (Travis and Kohn \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, for some plant species, the negative effects of increased HPT may be outweighed by the benefits of enhanced conspecific pollen transfer. Even pollinators carrying pollen from multiple species may still provide a net benefit to plants blooming in harsh environments (Gavini et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lopes et al. 2021). We should also note that groups with comparable levels of pollen species diversity, such as nocturnal moths and worker bumble bees, often exhibited marked differences in the combination of richness and evenness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The consequences of such variation in heterospecific pollen composition for plant reproductive success may depend not only on the total diversity of pollen grains but also on the phylogenetic relatedness among pollen donors (Arceo-G\u0026oacute;mez and Ashman \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Streher et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWith these caveats in mind regarding how different body pollen profiles affect plant reproduction, our findings offer several intriguing implications. The most striking is that when pollinator quality is reassessed based on the likelihood of causing HPT, a single conventionally recognized functional group may actually be divided into multiple groups. If this is the case, different phenotypes could evolve within a group of flowers classified as a 'bee-pollinated,' 'beetle-pollinated,' or 'fly-pollinated' system. For example, flowers frequently visited by pollinators with higher HPT propensity may be more likely to evolve floral traits that reduce the risk of HPT with closely related species blooming nearby\u0026mdash;such as unique floral colors or scents that promote individual specialization in foraging (Gumbert et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) or traits like prior selfing (Randle et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Katsuhara and Ushimaru \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In other cases, heterospecific pollen deposition may reduce opportunities for effective pollination by more faithful visitors, driving the evolution of filter traits that actively exclude low-fidelity pollinators (Ohashi et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, a 'bee-pollinated' flower may evolve traits that exclude solitary bees to minimize HPT while still allowing visits from bumble bees. In such cases, grouping all bees into a single functional group could obscure important evolutionary consequences, particularly those related to the evolution of floral filters.\u003c/p\u003e\u003cp\u003eIn sum, we demonstrated significant variation in heterospecificity of body pollen both among and within conventional functional groups of pollinators through an exhaustive comparison of pollen loads on individual insect bodies. Our findings highlight the need to place greater emphasis on the behavioral aspects of pollinators, alongside morphological traits, when defining their functional groups. Building on our current findings, a better understanding of how such diversity in pollen heterospecificity affects plant reproduction could contribute to a more comprehensive view of pollinator quality, their ecological significance, and, in turn, their evolutionary relationships with floral phenotypes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e- The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e- The authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e- All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e- The authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo formal ethical approval was required for this research, as it involved the study of insects, which are not subject to formal ethics approval processes in Japan.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are available on Figshare: https://figshare.com/s/83950d8b1bece4dbfd12 (Terada \u0026amp; Ohashi, 2025). Note: this is a private link; a DOI will be provided upon acceptance.\u003c/p\u003e\u003cp\u003e\u003cu\u003eAcknowledgments\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Yukie Sato and Mitsuru Hirota for their valuable advice and discussions. Tomoyuki Yokoi and his laboratory members provided opportunities for discussion. Shinichi Nagatani (NPO Tsukuba Environmental Forum) granted permission for fieldwork in \u003cem\u003eSusomi no Mori\u003c/em\u003e (2019\u0026ndash;2020). Aoi Nikkeshi provided guidance on pollen counting and species identification, and Saiki Fujii assisted with Lepidoptera identification. Minori Okubo and Marie Yamaguchi contributed to plant surveys and insect collection. Discussions with the members of the Ecological Interactions Laboratory at the University of Tsukuba further enriched this study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cu\u003eAuthor contributions\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eKohei Terada and Kazuharu Ohashi conceived the ideas, designed the methodology, and discussed the data analysis and interpretation. Kohei Terada led all fieldwork, laboratory sample processing, and data analyses. Both authors contributed substantially to the drafts and approved the final version for submission.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlarc\u0026oacute;n, R. (2010). Congruence between visitation and pollen-transport networks in a California plant-pollinator community. \u003cem\u003eOikos\u003c/em\u003e, \u003cem\u003e119\u003c/em\u003e(1), 35\u0026ndash;44. https://doi.org/10.1111/j.1600-0706.2009.17694.x\u003c/li\u003e\n\u003cli\u003eAlarc\u0026oacute;n, R., Waser, N. 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The topological differences between visitation and pollen transport networks: a comparison in species rich communities of the Himalaya\u0026ndash;Hengduan Mountains. \u003cem\u003eOikos\u003c/em\u003e, \u003cem\u003e128\u003c/em\u003e(4), 551\u0026ndash;562. https://doi.org/10.1111/oik.05262\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"},{"header":"Supplementary Files","content":"\u003cp\u003eSupplementary Files are not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Body pollen, Functional groups, Heterospecific pollen transfer, Pollen species diversity, Plant-animal interactions, Pollinators","lastPublishedDoi":"10.21203/rs.3.rs-7135148/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7135148/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSelection pressures exerted by pollinators are widely regarded as key drivers of the evolution of floral phenotypes. To capture presumed similarities in these selective forces, pollinators have traditionally been organized into functional groups, often corresponding to taxonomic classifications. However, given the likely diversity of foraging behavior among pollinators, groupings based solely on taxonomy or morphology may not adequately reflect variation in pollen transfer efficiency, particularly in terms of heterospecific pollen transfer (HPT) potential. To explore this possibility, we assessed how 11 groups of insect pollinators in a Japanese \u003cem\u003esatoyama\u003c/em\u003e landscape, classified by both taxonomy and putative behavioral differences, varied in the heterospecificity of pollen on their bodies. Despite being collected from flowers of the same \u003cem\u003eLigustrum\u003c/em\u003e trees in close proximity, the body pollen profiles of these insects significantly differed\u0026mdash;both among and within the 11 groups\u0026mdash;in terms of the proportion of heterospecific (non-\u003cem\u003eLigustrum\u003c/em\u003e) pollen, the composition of pollen species, and the diversity of pollen species. Notably, marked variations were found within bees, flies, and beetles, as well as between males and females of the same bumble-bee species. Based on these data, we discuss how the observed variations could offer new insights into conventional views on functional groups of pollinators, which are largely based on taxonomy or morphological/size similarities.\u003c/p\u003e","manuscriptTitle":"Heterospecificity of body pollen varies across and within functional groups of pollinators","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 22:57:32","doi":"10.21203/rs.3.rs-7135148/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd83c619-d95b-43d4-8596-c157a667a127","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T18:38:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 22:57:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7135148","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7135148","identity":"rs-7135148","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}