Main conclusionA hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detectedAbstractFerns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.