The vast abundance of terpene natural products in nature is due to enzymes known as terpene synthases (TPSs) that convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic carbon skeletons. Yet the evolution of TPSs is not well understood at higher levels of classification. Microbial TPSs from bacteria and fungi are only distantly related to typical plant TPSs, whereas genes similar to microbial TPS genes have been recently identified in the lycophyte Selaginella moellendorffii. The goal of this study was to investigate the distribution, evolution, and biochemical functions of microbial terpene synthase-like (MTPSL) genes in other plants. By analyzing the transcriptomes of 1,103 plant species ranging from green algae to flowering plants, putative MTPSL genes were identified predominantly from nonseed plants, including liverworts, mosses, hornworts, lycophytes, and monilophytes. Directed searching for MTPSL genes in the sequenced genomes of a wide range of seed plants confirmed their general absence in this group. Among themselves, MTPSL proteins from nonseed plants form four major groups, with two of these more closely related to bacterial TPSs and the other two to fungal TPSs. Two of the four groups contain a canonical aspartate-rich "DDxxD" motif. The third group has a "DDxxxD" motif, and the fourth group has only the first two "DD" conserved in this motif. Upon heterologous expression, representative members from each of the four groups displayed diverse catalytic functions as monoterpene and sesquiterpene synthases, suggesting these are important for terpene formation in nonseed plants.terpene synthase | specialized metabolism | nonseed plant | gene evolution T erpenoids are the largest class of land plant secondary metabolites, but they are not uniformly distributed in the plant kingdom (1). Many seed plants (angiosperms and gymnosperms) produce terpenoids of diverse types in large quantities. However, among nonseed plants, only liverworts are known as copious producers of terpenoids (2). Terpenoids have diverse biological and ecological functions with many serving as chemical defenses against herbivores and pathogens (3, 4). Some have lineagespecific functions, such as the volatile terpenoids in flowers that are involved in attracting pollinators (5). Characterizing the biosynthesis of terpenoids in all plant lineages is therefore an important avenue to understanding their roles in the adaptation of various lineages of terrestrial plants.Terpene synthases (TPSs) are pivotal enzymes for terpenoid biosynthesis, forming a distinctive superfamily based on both sequence identity and structure classification. However, within this group, typical plant and microbial (bacterial and fungal) TPSs share very low sequence similarity and are therefore only distantly related (6). The typical plant TPSs form subfamilies with individual subfamilies generally associated with specific biochemical functions, such as monoterpene, sesquiterpene, or diterpene biosynthesis (7,8). Monoterpene synthases a...