Huperzia species vary greatly in Lycopodium alkaloid composition and content. ► Controlled growth conditions are important for evaluating alkaloid variation. ► Genetics and the environment play important roles in controlling HupA production. ► H. pinifolia switched from HupA to nankakurine production when callus was induced. ► UPLC–IMS-TOFMS is ideally suited for rapid quantification of Lycopodium alkaloids.