Abstract
Lophophora diffusa is a psychoactive plant of Cactaceae, morphologically similar to the mescaline-rich Lophophora williamsii but with a distinct alkaloid profile, posing significant challenges for law enforcement. Here, we sequenced the complete chloroplast genome of L. diffusa using PacBio high-throughput sequencing technology. The chloroplast genome is 115,689 bp in length and exhibits a highly derived quadripartite structure, featuring a large single-copy (LSC) region of 79,403 bp, a small single-copy (SSC) region of 32,910 bp, and a pair of significantly reduced inverted repeat (IR) regions of 1,688 bp each. It encodes 100 unique genes and has a GC content of 36.52%. Phylogenetic analysis robustly resolved the position of L. diffusa, formed a monophyletic Lophophora clade that includes L. fricii and L. williamsii; under the current sampling, this clade was resolved as sister to a group containing Mammillaria species. This study provides a foundational genomic resource for future development of precise molecular tools to distinguish L. williamsii from its relatives, thereby supporting forensic botany and international narcotics control efforts.