Floral scent is crucial for mediating plant-pollinator interactions, which are one of the main drivers of diversification in angiosperms. Floral scent is a dynamic phenotype, which can be fine-tuned in both content and emission rate to suit the behavioural patterns of specific pollinators. Asarum sect. Heterotropa (Aristolochiaceae) is one of the most highly diversified plant lineages in Japan and exhibits remarkable variation in floral scent across the phylogeny. This is hypothesised to be part of a floral mimicry strategy to attract specific pollinators, which has likely contributed to the diversification of this lineage. This study sought to characterise the floral scents and pollination systems across Heterotropa, which remain largely undescribed.
We have tentatively identified pollinators for eight Heterotropa species, which were mainly phorid flies (Phoridae), fungus gnats (Mycetophilidae) and small dung flies (Sphaeroceridae), and were specific to each Heterotropa species. We characterised the floral scents by headspace-solid phase microextraction (SPME) sampling and gas chromatography-mass spectrometry (GC-MS) analysis and identified the main volatile classes as oligosulphides, terpenes, alcohols and esters, with great variation in scent profiles across the phylogeny. Given that the pollinators we observed in the field visited flowers almost exclusively during the daytime, we analysed scent emission patterns by time-course GC-MS and real time proton transfer reaction-mass spectrometry (PTR-MS). We found that the dominant floral volatiles are emitted in a diurnal pattern that corresponds to the pollinator visitation time we observed. By quantifying the degree of overlap between the volatile emission and visits by different animals, we showed that floral volatile emission is synchronised exclusively with pollinator visits. As both the floral volatiles and the pollinators vary across Heterotropa species, these results suggest that floral scent has evolved as a fine-tuned adaptive trait to attract specific pollinators, which has likely played an important role in the diversification of this plant lineage.