An early-diverging Caladeniinae orchid reveals diversification of terpene and apocarotenoid pathways underlying floral scent evolution

Orchids exhibit extraordinary floral diversity, yet the molecular mechanisms underlying floral scent evolution remain poorly understood. Guided by recent phylogenomic reconstruction of the Australian orchid subtribe Caladeniinae, we used Glossodia major, an earlier-diverging food-deceptive lineage, as an evolutionary anchor for investigating floral volatile diversification. Scent-producing petals showed coordinated expression of terpene synthase (TPS), carotenoid and carotenoid cleavage dioxygenase (CCD) pathways. Functional characterisation identified GmTPS-b3, GmTPS-b4 and GmTPS-a1 as contributors to floral monoterpene and sesquiterpene production, while GmCCD1 produced geranylacetone, the dominant floral volatile, from {zeta}-carotene. GmCCD4 and GmCCD7a showed additional, partially overlapping apocarotenoid-forming activities. Integration of tissue-specific expression, enzyme activity and subcellular localisation linked these pathways to the emitted floral bouquet. Functional comparisons with characterised Caladenia homologues revealed biochemical conservation and catalytic and compartmental specialisation, while expression profiling across 31 species identified pollination strategy-associated regulatory divergence in TPSa1 and TPSb3, consistent with corresponding shifts in terpenoid-rich floral scent. We propose that differential modification and deployment of conserved biosynthetic pathways contributed to the attenuation and re-emergence of terpenoid-rich floral scent during Caladeniinae diversification. More broadly, our findings provide candidate mechanistic links between molecular evolution, pollinator-mediated selection and floral diversification.