Polyploidy has played a key role in plant evolution and diversification. Despite this, the processes governing reproductive isolation among cytotypes growing in mixedploidy populations are still largely unknown. Theoretically, coexistence of diploid and polyploid individuals in sympatric populations is unlikely unless cytotypes are prezygotically isolated through assortative pollination. Here, we investigated the pre-mating barriers involved in the maintenance of three co-occurring cytotypes from the genus Gymnadenia (Orchidaceae): tetraploid and octoploid G. conopsea and tetraploid G. densiflora. We assessed differences in flowering phenology, floral morphology, and visual and olfactory cues, which could lead to assortative mating. Gas chromatography coupled with Electronic supplementary material The online version of this article (electroantennographic detection was used to identify scent compounds with physiological activity in the two main pollinators, Deilephila porcellus and Autographa gamma. The importance of olfactory cues was also assessed in the field by analysing the moths' responses to the olfactory display of the plants, and by following the pollinator's behaviour on artificial arrays. Our complex approach demonstrated that the coexistence of Gymnadenia cytotypes in mixed-ploidy populations was only partly explained by differences in floral phenology, as cytotypes with overlapping flowering (i.e., octoploid G. conopsea and tetraploid G. densiflora) might freely exchange pollen due to only 1 mm differences in spur lengths and the lack of assortative behaviour of pollinators. While floral colour among the cytotypes was similar, floral scent differed significantly. Though both pollinator species seemed to physiologically detect these differences, and the floral scent alone was sufficient to attract them, pollinators did not use this cue to discriminate the cytotypes in the field. The absence of premating barriers among cytotypes, except partial temporal segregation, suggests the existence of other mechanisms involved in the cytotypes' coexistence. The genetic differences in ITS sequences among cytotypes were used to discuss the cytotype's origin.

Many animals feed on flowers, and visual as well as olfactory cues are considered as most important mediators in animal-plant interactions. However, the relative importance of these cues is not well understood. Bees are the most important animal pollinators worldwide and here, we determined the importance of decoupled and combined visual and olfactory cues of Lysimachia punctata (Primulaceae) for host plant location in both sexes of the specialized, solitary bee, Macropis fulvipes (Melittidae). Lysimachia-inexperienced female bees preferred olfactory over visual cues though visual cues increased the attractiveness of olfactory ones. In experienced females, the importance of visual cues was increased. Both Lysimachia-naive and -experienced males relied more on visual cues as compared to females. This study demonstrates that the relative weighting of cues used for host plant finding depends on the sex and experience of M. fulvipes. The latter finding reveals the presence of learning-induced behavioural plasticity in host plant finding for a bee species. It may allow the bee to forage highly efficient. Visually guided female detection on flowers by males is a likely functional explanation for the differences in the weighting of visual and olfactory cues between the sexes.

Honeybee toxicology is complex because effects on individual bees are modulated by social interactions between colony members. In the present study, we applied high doses of the insect growth regulator fenoxycarb to honeybee colonies to elucidate a possible interplay of individually- and colony-mediated effects regarding honey bee toxicology. Additionally, possible effects of the solvent dimethyl sulfoxide (DMSO) were assessed. We conducted studies on egg hatching and brood development to assess brood care by nurse bees as well as queen viability. Egg hatching was determined by the eclosion rate of larvae from eggs originating from colonies (i) treated with sugar syrup only, (ii) treated with sugar syrup containing DMSO and (iii) treated with sugar syrup containing fenoxycarb (dissolved in DMSO). To evaluate brood development, combs with freshly laid eggs were reciprocally transferred between colonies, and development of brood was examined in the recipient hive. Brood reared inside DMSO- and fenoxycarb-treated colonies as well as brood from DMSO- and from fenoxycarb-exposed queens showed higher mortality than brood not exposed to the chemicals. No differences were found in egg hatching among the treatments, but there was a higher variability of eclosion rates after queens were exposed to fenoxycarb. We also observed queen loss and absconding of whole colonies. Based on our results we infer that fenoxycarb has queen- as well as nurse bee-mediated effects on brood quality and development which can lead to the queen's death. There also is an effect of DMSO on the nurse bees' performance that could disturb the colony's equilibrium, at least for a delimited timespan.

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