Floral scent diversity and evolution in deceptive Cypripedium, and post-pollination effects on floral scent and its biosynthesis in C. henryi

Background and Aims: Floral scent is a primary driver of pollinator attraction and reproductive isolation in deceptive orchids. Yet, little is known about the variation and evolution of scent in a phylogenetic framework and how pollination affects the biosynthesis and emission of scents in these lineages. This study characterizes scent diversity across Cypripedium, a genus of deceptive orchids, examines phylogenetic constraints, and investigates post-pollination effects on scent composition in one of the species. Methods: Floral volatile organic compounds (VOCs) were sampled from 31 Cypripedium taxa using dynamic headspace techniques, and analysed by gas chromatography coupled to mass spectrometry (GC/MS). Uni- and multivariate methods were used to analyse intra- and interspecific scent variation and test for phylogenetic signal in floral scent. Post-pollination effects on scent composition were experimentally tested in C. henryi using four pollination treatments, combined with RNA-seq analyses of floral tissues to examine transcriptional responses in VOC biosynthetic pathways. Key results: We detected 258 floral VOCs, revealing a high chemical diversity dominated by terpenoids, aliphatics, and aromatic compounds. Floral scent composition was highly species-specific, and closely related taxa often shared only few compounds. Scent composition differed among pollination groups, but these groups explained only a limited proportion of overall scent variation. We detected a significant but weak phylogenetic signal, suggesting limited evolutionary constraint in floral scent. Pollination had limited effect on C. henryis floral scent and volatile biosynthesis gene expression, despite a significant decrease in total scent emission after pollinium deposition. Transcriptional changes predominantly occurred in the petals rather than the labellum and staminode. Conclusion: Floral scent evolution in Cypripedium is characterized by high chemical diversity, and a weak phylogenetic constraint, consistent with variable selection in food-deceptive systems. The pollination study further suggests the absence of a strong, directed post-pollination effect on the floral scent of C. henryi. Keywords: Cypripedium; deceptive pollination; floral scent; volatile organic compounds (VOCs); phylogenetic signal; linalool; post-pollination effects; transcriptome profiles; differential gene expression; floral organs