Putative Pollinators Research Articles

Morphological and palaeoecological aspects of fossil insects unveiled by UV-A light

Studying insect fossils, particularly those preserved as compressions in sedimentary matrices, can be difficult due to the taphonomic processes that often result to poor preservation and contrast of structures compared to the embedding matrix. To address this, we propose a user-friendly and simple methodology based on UV-light to study insect fossils and select specimens of interest for more advanced imagery exploration. While UV-light imaging has been previously applied to compressions of arthropod fossils, it typically involved laser light sources. Our approach allows the investigation of fossils using an affordable, compact, and portable UV-light source, along with a simple and replicable low-cost protocol.•The methodology is based on UV-light induced natural fluorescence of sediment and fossil remains.•UV-light is effective on compression fossils to gain natural contrast and enhance observation of body structures like veins or setae on wings.•UV-light is effective to reveal palaeoecological information such as pollen grains preserved on specimens, especially near or on putative pollinator or pollen-eating taxa.

Near extinct Argyreia versicolor and rare Argyreia mekongensis are dependent on carpenter bee pollinators.

Argyreia versicolor and Argyreia mekongensis are extremely rare plant species. The former had not been seen for nearly 100 years until two individuals were found in Thailand in 2018, and only a handful of populations are known for the latter. The aims of this study were to examine the breeding systems of A. versicolor and A. mekongensis using pollination experiments and to determine their potential pollinators via floral observations. Our controlled pollination experiments uncovered the self-incompatibility of both species. Pollinator censuses indicated that females of two carpenter bee species, Xylocopa aestuans and Xylocopa latipes, were the predominant floral visitors for both Argyreia species. Our observations confirmed a harmonious match between the floral shape of both Argyreia species and the body sizes of these pollinators, ensuring effective pollen transfer and validating their role as putative pollinators. In line with the high frequency of pollinator visits observed, our controlled pollination experiments found no evidence of pollen limitation under field conditions. The findings of this study hold significance for the conservation of these endangered species, yet the situation is dire for A. versicolor, with one of the two individuals under study recently lost. Hence, it is crucial to intensify monitoring efforts for the species, aiming to identify additional individuals for potential inclusion in an ex-situ conservation program. Simultaneously, safeguarding the habitat of these plant species and their pollinators will be critical.

Floral ecology of Puya ctenorhyncha (Bromeliaceae) an endemic plant of Bolivia

Background: The terrestrial bromeliad Puya ctenorhyncha is a near-threatened endemic species that grows between 2,500-4,050 m asl.
 Hypotheses: Hummingbirds are the most important visitors of this plant given its morphological and floral traits.
 Study site: The study was carried out along the “Death Road”, located in the Yungas montane cloud forest, La Paz, Bolivia.
 Methods: We monitored an average of 216 individuals monthly for two years (2018-2019). Besides we conducted direct observations of focal plants to quantify the visits of animals.
 Results: The reproductive season lasted from April to August in the first year and from May to June in the second. The mean nectar volume was 8.09 ± 1.59 μL per flower and its composition included fructose, glucose, and 86 % of water. With 341 hours of observations, we determined that Coeligena torquata (Trochilidae) is the main visitor and putative pollinator, followed by C. violifer. Males of both species were more frequent visitors than females. Coeligena torquatashowed the highest activity during the afternoon while C. violifer was more active in the morning.
 Conclusions: Floral phenology seems to be annual and unimodal. Although the main pollinators seem to be hummingbirds, the nectar concentration and composition is more similar to bromeliad species of other genera which are pollinated by bats or passerine birds. It is important to evaluate, in future studies, the dependence of both hummingbirds and their behavioural response towards the plant.

3D pollination biology using micro-computed tomography and geometric morphometrics in Theobroma cacao.

Imaging technologies that capture three-dimensional (3D) variation in floral morphology at micro- and nano-resolutions are increasingly accessible. In herkogamous flowers, such as those of Theobroma cacao, structural barriers between anthers and stigmas represent bottlenecks that restrict pollinator size and access to reproductive organs. To study the unresolved pollination biology of cacao, we present a novel application of micro-computed tomography (micro-CT) using floral dimensions to quantify pollinator functional size limits. We generated micro-CT data sets from field-collected flowers and museum specimens of potential pollinators. To compare floral variation, we used 3D Slicer to place landmarks on the surface models and performed a geometric morphometric (GMM) analysis using geomorph R. We identified the petal side door (an opening between the petal hoods and filament) as the main bottleneck for pollinator access. We compared its mean dimensions with proposed pollinators to identify viable candidates. We identified three levels of likelihood for putative pollinators based on the number of morphological (body) dimensions that fit through the petal side door. We also found floral reward microstructures whose presence and location were previously unclear. Using micro-CT and GMM to study the 3D pollination biology of cacao provides new evidence for predicting unknown pollinators. Incorporating geometry and floral rewards will strengthen plant-pollinator trait matching models for cacao and other species.

Possible long-proboscid insect pollinators from the Early Permian of Russia

Insect pollination is one of the hallmarks of flowering plants.1 Bees, moths, flies, and some other pollinators evolved elongate siphonate mouthparts for sucking concealed nectar and occasionally other liquids.2 However, it is clear from the fossil record that insects with similar adaptations appeared long before the mid-Cretaceous radiation of angiosperms. These insects most probably used their proboscis to reach pollination drops and other sugary fluids that were hidden in the cones of extinct gymnosperms, pollinating them in the process.3-6 The vast majority of these gymnosperm-associated long-proboscid insects have been reported from the Middle Jurassic to the Early Cretaceous, i.e., the time interval that immediately predated the advent of flowering plants.7 By contrast, the Paleozoic stage of the co-evolution between long-proboscid insect pollinators and plants has remained poorly understood. Here, we report a putative pollination mutualism involving long-proboscid holometabolous insects (Panorpida: Protomeropidae) from the Early Permian of Russia (ca. 283-273 Ma). Their elongate mouthparts have very similar morphology to those of some present-day nectarivorous Coleoptera and Hymenoptera and probably served to imbibe micropylar secretions from the semi-closed ovulate organs of the gymnosperms of a peltaspermalean affinity that have been found in the same locality. This is the earliest record of insects with siphonate-like mouthparts, which could indicate that the complex interactions between pollinators and gymnosperms predate the first flowering plants by over 100 Ma.

Native bees as putative pollinators of the avocado Persea americana Mill. cv. Hass in Colombia

Hymenoptera, especially bees, play a fundamental role in increasing the fruitiness and quality of the avocado through proper pollination. This study proposed to determine hymenopteran floral visitor fauna and potential pollinators of the Hass avocado cultivar in Colombia. The research was carried out on a single plot of land on a farm during two consecutive flowering periods (Aug-Sep. 2013/ Feb-Mar. 2014), randomly selecting four trees for the sampling and changing them every week. Hymenoptera richness did not differ significantly between flowering periods. Besides Apis mellifera, a wide diversity of floral visitors was found in the cultivar studied. Thirteen families of Hymenoptera were recorded, among them, three bee species of the Meliponini tribe: Scaptotrigona barrocoloradensis (Schwarz) (37%), Partamona cf. aequatoriana Camargo (32%) and Tetragonisca angustula (Latreille) (17.65%). Bees carried pollen from 18 botanical families, the most frequent were: Asteraceae (30.08%), Lauraceae (28.02%) and Urticaceae (19.49%). Although A. mellifera has the highest diversity and abundance in its pollen load, the stingless bees together transported about half of the available avocado pollen (47.53%). These bees stood out for having greater specificity in their pollen load, on surveying the insect body areas where they transport pollen. In A. mellifera, P. cf. aequatoriana and S. barrocoloradensis, avocado pollen adheres mainly to the legs while in T. angustula the greater pollen adherence was in the head and thorax. Although A. mellifera was the main pollinator, as a result of greater visit frequency and pollen load, the potential importance of the native bee species for pollination services is discussed.

Reproductive biology of Syagrus coronata (Arecaceae): sex-biased insect visitation and the unusual case of scent emission by peduncular bracts.

Several monoecious species of palms have developed complex strategies to promote cross-pollination, including the production of large quantities of floral resources and the emission of scents that are attractive to pollinators. Syagrus coronata constitutes an interesting model with which to understand the evolution of plant reproductive strategies in a monoecious species adapted to seasonally dry forests. We monitored blooming phenology over 1year, during which we also collected and identified floral visitors and putative pollinators. We identified potential floral visitor attractants by characterizing the scent composition of inflorescences as well as of peduncular bracts, during both male and female phases, and the potential for floral thermogenesis. Syagrus coronata produces floral resources throughout the year. Its inflorescences are predominantly visited by a diverse assortment of small-sized beetles, whose richness and abundance vary throughout the different phases of anthesis. We did not find evidence of floral thermogenesis. A total of 23 volatile compounds were identified in the scent emitted by the inflorescences, which did not differ between male and female phases; whereas the scent of the peduncular bracts was composed of only 4-methyl guaiacol, which was absent in inflorescences. The composition of floral scent chemistry indicates that this palm has evolved strategies to be predominantly pollinated by small-sized weevils. Our study provides rare evidence of a non-floral scent emitting structure involved in pollinator attraction, only the second such case specifically in palms. The peculiarities of the reproductive strategy of S. coronata might play an important role in the maintenance of pollination services and pollen dispersion.

Pollination biology reveals challenges to restoring populations of Brighamia insignis (Campanulaceae), a critically endangered plant species from Hawai‘i

Understanding the reproductive biology of rare plant species is fundamental to managing their restoration. Brighamia insignis is a critically endangered Hawaiian lobeliad endemic to the islands of Kauaʻi and Niʻihau. The flowers appear to be adapted for moth pollination although its putative pollinator is believed to be extinct or very rare. To confirm the pollination syndrome, document the breeding system, and identify potential pollinators of B. insignis: 1) a suite of floral characters were examined, 2) pollination treatments were performed, and 3) diurnal and nocturnal floral visitor observations were conducted at an ex situ site on Kaua‘i. Brighamia insignis flowers contain sucrose-rich nectar and emit a strong floral scent containing benzyl alcohol, linalool, and methyl salicylate. Pollination treatments revealed that the species is primarily outcrossing with several of the study plants also capable of low levels of selfing. However, most of the plants had low pollen production and viability. No moths and only occasional non-native insect species visited the flowers of B. insignis. None of these insect visitors appeared to be serving as effective pollinators. In spite of this lack of flower visitation by moths, analysis of nectar and floral scent support a moth pollination syndrome in B. insignis. The potential loss of pollinators suggests that restoring populations of B. insignis may not be feasible; human assisted cross-pollination would be necessary for fruit and seed to set, as only 1% of the control and self treatment flowers formed fruit. Therefore, this species appears dependent upon intense human management to prevent its extinction.

A new species of Pleurothallis (Orchidaceae: Pleurothallidinae) from Valle del Cauca, Colombia and a note on the relationship between subsections Macrophyllae-Racemosae and Antenniferae

A new species of Pleurothallis from subsection Macrophyllae-Racemosae is described from Valle del Cauca, Colombia. Morphological characteristics to distinguish this species from Pleurothallis bicornis, P. subreniformis and P. torrana are presented. The micromorphology of the lip of the new species was studied utilizing scanning electron microscopy. Labellar micromorphology is discussed in relation to putative pollination mechanisms.

Paternity analysis in a ‘Rainier’ open pollination population usingS-alleles and microsatellite genotyping

Fruit breeding programs usually use controlled hand pollination among cultivars and advanced selections for obtaining segregating populations to select new cultivars. In sweet cherries, however, sometimes in controlled pollination few hybrids are obtained. Caging whole trees with bees and flowers of the pollinating cultivar is sometimes used to obtain larger hybrid populations. To generate large segregating populations for the Chilean Sweet Cherry Breeding Program (run by the Consortium of BioFrutales S.A. and Instituto de Investigaciones Agropecuarias), the initial strategy was to harvest seeds from open pollinated self-incompatible cultivars maintained at the germplasm collection orchard of Univiveros, one of the leading fruit tree nurseries in Chile. While the female parent is known, the male parent is unknown. The pollen source will depend on the cultivars present in the area, the flowering time and the compatibility of the cultivars involved. In order to identify pollinators of the self-incompatible 'Rainier', which is surrounded by several other cultivars in the collection, a group of them were proposed as the most possible pollen donors considering their flowering time and location within the orchard. An initial screening using S-allele genotyping of 675 one-year-old seedlings from 'Rainier' open-pollination allowed for the identification of several S-genotypes, which suggested putative pollinators. These analyses identified two main putative pollen donors: 'Rivedel' (232 individuals, 34% of total) and 'Vanda' (101 individuals, 15% of the total). The paternity of these cultivars was confirmed or rejected after additional analysis with at least five highly informative microsatellite markers. The segregating population resulting from the cross 'Rainier' × 'Rivedel' (n=232) is currently being analyzed with microsatellite markers in order to build a linkage map for sweet cherry. In this population, parents exhibit contrasting phenotypes for skin and flesh color and berry firmness, among other agronomically important traits, making this population very interesting for future genetic studies.

Floral micromorphology of the genus Restrepia (Orchidaceae) and the potential consequences for pollination

Restrepia is a small Pleurothallid genus, comprising 57 species, 44 of which were discovered since 1970. These species are indigenous to Central and South America, where their montane forest habitats are under increasing pressure from changes in land use. With resulting increasingly fragmented habitats and dwindling numbers, the pollination systems of obligate out-breeding genera, such as Restrepia, may no longer function efficiently which could potentially lead to their extinction. As such, the main aim of the current study was to perform an in-depth investigation of floral structures in the genus, using SEM and photographic technology to formulate a putative pollination mechanism for these species.The floral micromorphology of dorsal sepal and lateral petal osmophores, synsepal, labellum, cirrhi and calli were investigated by scanning electron microscopy (SEM), macro-photography and quantitative analyses of some floral proportions.The secretory nature of the labellum, synsepal and osmophore papillae were established and the calli were shown to possess a unique papillate, non-secretory structure. A pollination mechanism for the genus was proposed which includes the role of the scent trails produced by the osmophores and the ‘trapping’ role of the cirrhi. A ‘functional fit’ between the flower and the pollinator is suggested. In conclusion, we consider Restrepia to represent a non-nectar rewarding and ‘deceptive’ orchid genus and that this pollination mechanism may be directly linked to the breeding system (gametophytic self-incompatibility) in this genus.

Petunia floral trait plasticity in response to soil nitrogen content and subsequent impacts on insect visitation

Plastic changes in floral trait phenotypes in response to biotic factors (i.e., herbivory and pollination) are known to impact pollinator behavior. In contrast, research on abiotic factors suggests substantial plasticity in floral traits, but has rarely explored the implications that such changes have on the behavior of putative pollinators. We explored the possibility of floral trait plasticity in Petunia hybrida Vilm. genotypes in response to soil nitrogen concentration, and the potential impact that such plasticity might have on insect visitation. Given the role of nitrogen in plant growth and biochemistry, we predicted increases in floral scent, pigmentation, and floral size with increased nitrogen. We further predicted that insect visitors would respond to this phenotypic plasticity by altering behavior patterns. Our results suggested little plasticity in floral color and floral scent in response to nitrogen. We found no changes in anthocyanin concentration per unit of floral tissue under increased nitrogen conditions. Of nine benzenoid/phenylpropanoid volatile compounds examined, only one (eugenol) increased per unit floral tissue with increasing nitrogen concentration. In contrast, floral size followed the predicted pattern, with clear increases in both corolla size and display size with increasing nitrogen. All traits varied among genotypes, indicating genotype-specific trait expression. Field-based choice experiments using genotypes from control, low, and high nitrogen treatments suggested support for our behavioral hypothesis: insects visited plants with increased soil nitrogen at a significantly higher rate than those without additional nitrogen. This pattern suggests that putative pollinators are likely responding to plastic changes in display size, corolla size, and/or eugenol emission. We also found a significant difference in insect visitation between genotypes, as predicted by floral phenotype differences found for these true-breeding lines, and a strong interaction between genotype and treatment, suggesting that differences in genotype responses to nitrogen treatments result in differential insect behavior patterns.

Fire effects on pollination in a sexually deceptive orchid

Research into the effectiveness of prescribed fire in managing pollination has only recently begun. The effects of fire on pollination have not been explored in sexually deceptive systems. Further, the potential for multiple effects operating at different spatial scales has not been explored in any pollination system despite multiscale effects on pollination observed in agricultural landscapes. We observed the frequency of pollinator visitation to flowers of sexually deceptive Caladenia tentaculata and related it to the post-fire age class of the vegetation at local and landscape scales. We also related the number of the pollinator’s putative larval hosts (scarab beetles) captured at these sites to age class. At the local scale (i.e. the sample location), visitation was highest in recently burnt sites. At the landscape scale, positive associations were observed between (1) putative pollinator hosts and vegetation burnt 36–50 years ago, and (2) pollinator visitation and vegetation burnt ≥50 years ago. Local- and landscape-scale effects on visitation were synergistic, such that visitation was greatest when fire age was heterogeneous within pollinator foraging range.

A novel reliable method of DNA extraction from olive oil suitable for molecular traceability

Extra virgin olive oil production has a worldwide economic impact. The use of this brand, however, is of great concern to Institutions and private industries because of the increasing number of fraud and adulteration attempts to the market products. Here, we present a novel, reliable and not expensive method for extracting the DNA from commercial virgin and extra virgin olive oils. The DNA is stable overtime and amenable for molecular analyses; in fact, by carrying out simple sequence repeats (SSRs) markers analysis, we characterise the genetic profile of monovarietal olive oils. By comparing the oil-derived pattern with that of the corresponding tree, we can unambiguously identify four cultivars from Samnium, a region of Southern Italy, and distinguish them from reference and more widely used varieties. Through a parentage statistical analysis, we also identify the putative pollinators, establishing an unprecedented and powerful tool for olive oil traceability.

The floral scents of Nymphaea subg. Hydrocallis (Nymphaeaceae), the New World night-blooming water lilies, and their relation with putative pollinators

Night-blooming water lilies are characterized by intense emission of floral VOCs. Their unique scent-oriented pollinators, cyclocephaline scarabs (Scarabaeidae, Cyclocephalini), are attracted to flowers that they use as reliable sources of food and as mating aggregation sites. Chemical analysis of floral scent samples of seven species of Nymphaea subg. Hydrocallis established remarkably simple fragrant blends, each of which was dominated by one or two prominent compounds that alone accounted for over 95% of total scent emission. A total of 22 VOCs were identified: aliphatics (9), C5-branched chain compounds (5) and aromatics (8). Anisole was the dominant constituent in the floral scents of Nymphaea amazonum subsp. amazonum, N. amazonum subsp. pedersenii and N. tenerinervia, whereas (methoxymethyl)benzene was the most abundant VOC in samples of N. lasiophylla and N. lingulata. Flowers of N. rudgeana and N. gardneriana emitted high amounts of methyl hexanoate and methyl 2-methylbutanoate. Comparisons of floral VOC composition including other day- and night-blooming species of Nymphaea and Victoria obtained from the literature evidenced disparities related to habitus. While flowers of day-blooming species mostly emit aromatic alcohols and ethers, nocturnal species are particularly rich in aromatic ethers, aliphatic esters and C5-branched chain esters. These findings strongly suggest that the floral scent composition within closely related Nymphaea and Victoria is linked to pollinator selection, and the putative role of floral VOCs in pollinator attractiveness is discussed.

Non-Density Dependent Pollen Dispersal of Shorea maxwelliana (Dipterocarpaceae) Revealed by a Bayesian Mating Model Based on Paternity Analysis in Two Synchronized Flowering Seasons

Pollinator syndrome is one of the most important determinants regulating pollen dispersal in tropical tree species. It has been widely accepted that the reproduction of tropical forest species, especially dipterocarps that rely on insects with weak flight for their pollination, is positively density-dependent. However differences in pollinator syndrome should affect pollen dispersal patterns and, consequently, influence genetic diversity via the mating process. We examined the pollen dispersal pattern and mating system of Shorea maxwelliana, the flowers of which are larger than those of Shorea species belonging to section Mutica which are thought to be pollinated by thrips (weak flyers). A Bayesian mating model based on the paternity of seeds collected from mother trees during sporadic and mass flowering events revealed that the estimated pollen dispersal kernel and average pollen dispersal distance were similar for both flowering events. This evidence suggests that the putative pollinators – small beetles and weevils – effectively contribute to pollen dispersal and help to maintain a high outcrossing rate even during sporadic flowering events. However, the reduction in pollen donors during a sporadic event results in a reduction in effective pollen donors, which should lead to lower genetic diversity in the next generation derived from seeds produced during such an event. Although sporadic flowering has been considered less effective for outcrossing in Shorea species that depend on thrips for their pollination, effective pollen dispersal by the small beetles and weevils ensures outcrossing during periods of low flowering tree density, as occurs in a sporadic flowering event.

Matching floral and pollinator traits through guild convergence and pollinator ecotype formation.

Pollinator landscapes, as determined by pollinator morphology/behaviour, can vary inter- or intraspecifically, imposing divergent selective pressures and leading to geographically divergent floral ecotypes. Assemblages of plants pollinated by the same pollinator (pollinator guilds) should exhibit convergence of floral traits because they are exposed to similar selective pressures. Both convergence and the formation of pollination ecotypes should lead to matching of traits among plants and their pollinators. We examined 17 floral guild members pollinated in all or part of their range by Prosoeca longipennis, a long-proboscid fly with geographic variation in tongue length. Attractive floral traits such as colour, and nectar properties were recorded in populations across the range of each species. The length of floral reproductive parts, a mechanical fit trait, was recorded in each population to assess possible correlation with the mouthparts of the local pollinator. A multiple regression analysis was used to determine whether pollinators or abiotic factors provided the best explanation for variation in floral traits, and pollinator shifts were recorded in extralimital guild member populations. Nine of the 17 species were visited by alternative pollinator species in other parts of their ranges, and these displayed differences in mechanical fit and attractive traits, suggesting putative pollination ecotypes. Plants pollinated by P. longipennis were similar in colour throughout the pollinator range. Tube length of floral guild members co-varied with the proboscis length of P. longipennis. Pollinator shifts have resulted in geographically divergent pollinator ecotypes across the ranges of several guild members. However, within sites, unrelated plants pollinated by P. longipennis are similar in the length of their floral parts, most probably as a result of convergent evolution in response to pollinator morphology. Both of these lines of evidence suggest that pollinators play an important role in selecting for certain floral traits.

Epitypification and ecological notes for the Malagasy holoparasite Hydnora esculenta (Hydnoraceae)

The root holoparasite, Hydnora esculenta is a poorly known perennial herb described from southwestern Madagascar. The key character in the original diagnosis was floral dimorphism, a hermaphroditic form and an apparently thinner and longer sterile form. Other useful diagnostic characters are lacking. The type specimens consist only of slices of fruit, and lack important diagnostic characters, thus, an epitype is designated here. The original description is amplified based on field collections and examination of available herbarium material. Flowers of H. esculenta are variable in size (11.6–26.9 cm long) and merosity (3–6), but are uniformly hermaphroditic like all other Hydnoraceae. Osmophores situated on tepal apices combined with angular rhizomes separate H. esculenta from other known Hydnora spp. Additionally, ribbed tepal margins for H. esculenta are diagnostic and are preserved on herbarium specimens. Pollination biology follows the general pattern for the genus, brood‐site mimicry with insect imprisonment and release. Floral mobbing events by the putative pollinator, a scavenger scarab beetle (Kuijtenous laviceps) are reported. The most common observed host of H. esculenta was the invasive tree Pithocellobium dulce. The range of H. esculenta maybe increasing due to the spread of P. dulce in riparian areas and disturbed habitats in southern Madagascar.

Shades of red: bird‐pollinated flowers target the specific colour discrimination abilities of avian vision

Colour signals are a major cue in putative pollination syndromes. There is evidence that the reflectance spectra of many flowers target the distinctive visual discrimination abilities of hymenopteran insects, but far less is known about bird-pollinated flowers. Birds are hypothesized to exert different selective pressures on floral colour compared with hymenopterans because of differences in their visual systems. We measured the floral reflectance spectra of 206 Australian angiosperm species whose floral visitors are known from direct observation rather than inferred from floral characteristics. We quantified the match between these spectra and the hue discrimination abilities of hymenopteran and avian vision, and analysed these metrics in a phylogenetically informed comparison of flowers in different pollination groups. We show that bird-visited flowers and insect-visited flowers differ significantly from each other in the chromatic cues they provide, and that the differences are concentrated near wavelengths of optimal colour discrimination by whichever class of pollinator visits the flowers. Our results indicate that angiosperms have evolved the spectral signals most likely to reinforce their pollinators' floral constancy (the tendency of individual pollinators to visit flowers of the same species) in communities of similarly coloured floral competitors.

The Lack of Pollinator Specificity in a Dioecious Fig Tree: Sympatric Fig-pollinating Wasps of Ficus septica in Southern Taiwan

Although species specificity between fig trees and their pollinators has been considered a classic example of obligate mutualism, increasing exceptions to the one-to-one relationship suggest that multiple pollinator species per fig host species may be a pervasive phenomenon. Based on both mitochondrial DNA and microsatellite analyses, we found three putative pollinator species (Ceratosolen spp.) associated with Ficus spetica in southern Taiwan, two of which, that differ in color, routinely coexist within single figs. These three pollinators are substantially divergent from the pollinator of F. spetica in New Guinea, implying that wasp diversity may be highly underestimated when the one-to-one rule is assumed. Our findings not only provide the first case of coexistence of pollinators within single figs on a shared dioecious Ficus host, but also an ideal system to investigate interspecific competition and sex allocation, especially when coexisting pollinators are visually distinguishable by their colors. TABLE S1. The number of Ceratosolen bisulcatus jucundus, C. sp. 1 and C. sp. 2 used in mitochondrial COI gene sequencing and microsatellite genotyping for each sampling locality and their sampling seasons. TABLE S2. Characteristics of seven microsatellite loci for six locations in southern Taiwan of Ceratosolen bisulcatus jucundus and C. sp. 1. FIGURE S1. FCA of three pollinators of Ficus septica in Taiwan based on seven microsatellite loci. A total of 560 wasps are included: 401 individuals of Ceratosolen sp. 1, 158 C. bisulcatus jucundus and one C. sp. 2. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.