In the sexually deceptive orchid genus

In sexually deceptive orchids, pollinator specificity is the main factor responsible for species isolation. The two sexually deceptive orchids Ophrys chestermanii and Ophrys normanii occur sympatrically on Sardinia and attract the same pollinator, males of Bombus vestalis, by producing the same odour bouquets. Since gene flow between these two species has been shown to be almost absent, their genetic distinctness seems to be preserved by other forms of reproductive barrier than pollinator specificity. The aim of this study is to investigate the nature and strength of these isolation barriers which apparently halt gene flow between these two orchids that share the same pollinator. Morphological measurements, combined with observations of cross-pseudocopulations, revealed that, in sympatry, the longer caudicles of O. normanii and the smaller and differently shaped stigmatic cavity in O. chestermanii are the main factors preventing gene flow between these two sympatric species. In contrast, other pre-or post-mating barriers seem to be weak or absent, as the two species have overlapping flowering time and we were able to rear hybrid offspring from interspecific manual crosses. At the same time, genetic analysis of orchid plantlets collected at sympatric sites detected the presence of only two putative hybrid individuals. Thus, the probability of natural cross-pollination Electronic supplementary material The online version of this article (

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Sexually deceptive orchids of the genus Ophrys attract male insects for pollination. Pollinator attraction is achieved by mimicking sex pheromones of virgin females of their pollinators, mostly bee species. In earlier investigations, we showed that the phylogenetically distinct Ophrys species O. chestermanii and O. normanii on Sardinia attract their pollinator, males of the cuckoo bumblebee B. vestalis, with the same bouquets of relatively polar volatile compounds. In this investigation, we studied the sex pheromone of virgin females of B. vestalis with the aim of identifying male-attracting compounds and of comparing them with labellum extracts of the two orchids, which were found to release male-attracting compounds in earlier investigations (Gögler et al. 2009). In bioassays, shock-frozen females, cuticle extracts and polar fractions of cuticle extracts of virgin females stimulated mating behaviour in the males. Using gas chromatography coupled with electroantennography (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS), we detected in polar fractions of cuticle extracts of B. vestalis females the same electrophysiologically active compounds as in labellum extracts of both orchid species, including aldehydes, esters, fatty acids and alcohols. Since statistical comparisons of the relative proportions of esters showed strong similarities between virgin females and orchids, our results support the hypotheses that this highly specialized Ophrys-pollinator relationship represents another case of chemical mimicry and that esters play a key role in male attraction.

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