The ways that plant-feeding insects have diversified are central to our understanding of terrestrial ecosystems. Obligate nursery pollination mutualisms provide highly relevant model systems of how plants and their insect associates have diversified and the over 800 species of fig trees (Ficus) allow comparative studies. Fig trees can have one or more pollinating fig wasp species (Agaonidae) that breed within their figs, but factors influencing their number remain to be established. In some widely distributed fig trees, the plants form populations isolated by large swathes of sea, and the different populations are pollinated by different wasp species. Other Ficus species with

Flower odor is often essential for attracting pollinators, and this is especially true for species-specific mutualism such as the one between Ficus species and their pollinating wasps. Receptive figs emit a stage and species specific odor composed of a mix of volatile organic compounds (VOC) that is attractive to their pollinators. Histological studies suggest that the ostiolar bracts are the main locus of fig floral VOC synthesis. To confirm a major role of the ostiole in VOC synthesis, detect the genes involved in VOC synthesis and analyze differences between closely related species, we compare the transcriptomes of pre-receptive and receptive figs of two closely related species, F. hirta and F. triloba. The two species presented similar numbers of expressed genes and similar annotation, classification results, and their upregulated unigenes belonged to similar biosynthetic pathways. However, phenylpropanoid and terpene synthesis were the main enriched VOC pathways in receptive figs of F. hirta while terpene and jasmonate synthesis were the main enriched VOC pathways in F. triloba. The shift in gene expression between pre-receptive and receptive figs was much more marked in the ostiolar bracts than in the flowers of F. hirta, and in the fig wall, confirming an important role of the ostiole in receptive fig odor emission. Only one unigene directly involved in VOC synthesis presented signatures of positive selection, further supporting that the source of interspecific receptive fig odor differentiation is based on gene regulation rather than on gene differentiation. Regulation based receptive fig odor differentiation between species suggests a potential for rapid evolution of this interspecific barrier

Background Host specificity among pollinator fig wasps (Agaonidae) depends on host plant specific volatile cues, but fig wasps must also pass through a narrow physical barrier (the ostiole) if they are to pollinate and oviposit. Across South East Asia the dioecious shrub Ficus hirta is associated with at least ten pollinator species allied to Valisia javana. Ficus triloba has a single recorded pollinator, Valisia esquirolianae. Receptive figs of F. hirta are usually much smaller than those of F. triloba, but at a mainland site where F. hirta has atypically large figs we identified both V. esquirolianae and V. javana from both Ficus species using COI and ITS2 sequencing. To investigate whether this host overlap was exceptional we reared fig wasps from the two trees elsewhere and recorded features that may facilitate host transfer between them, including attractant volatiles, reproductive phenology and the sizes of their figs and fig wasps. Results The two Ficus species were found to support both Valisia species at several of the sites, suggesting that the differences we detected in volatile profiles, ostiole sizes and pollinator head sizes are not strict barriers to host sharing. Valisia javana colonised F. triloba more frequently than V. esquirolianae colonised F. hirta. Conclusions This asymmetric sharing of pollinators may reflect the relative abundance of the two species of fig wasps and differences in host reproductive phenology. Asynchronous flowering of individual F. hirta may favor local retention of pollinators, in contrast to the tree-wide synchrony of F. triloba figs, which can generate local shortages of V. esquirolianae. If the pollinator sharing by male figs of F. triloba and F. hirta also occurs in female figs then this could result in gene flow between them.