Species of Osmanthus are economically important ornamental trees, yet information regarding their plastid genomes (plastomes) has rarely been reported, thus hindering taxonomic and evolutionary studies of this small but enigmatic genus. Here, we performed comparative genomics and evolutionary analyses on plastomes of 16 of the 28 currently accepted species, with 11 plastomes newly sequenced. Phylogenetic studies identified four main lineages within the genus that are here designated: ‘Caucasian Osmanthus’ (corresponding to O. decorus), ‘Siphosmanthus’ (corresponding to O. sect. Siphosmanthus), ‘O. serrulatus + O. yunnanensis’, and ‘Core Osmanthus’ (corresponding to O. sect. Osmanthus + O. sect. Linocieroides). Molecular clock analysis suggested that Osmanthus split from its sister clade c. 15.83 Ma. The estimated crown ages of the lineages were the following: genus Osmanthus at 12.66 Ma; ‘Siphosmanthus’ clade at 5.85 Ma; ‘O. serrulatus + O. yunnanensis’ at 4.89 Ma; ‘Core Osmanthus’ clade at 6.2 Ma. Ancestral state reconstructions and trait mapping showed that ancestors of Osmanthus were spring-flowering and originated at lower elevations. Phylogenetic principal component analysis clearly distinguished spring-flowering species from autumn-flowering species, suggesting that flowering time differentiation is related to the difference in ecological niches. Nucleotide substitution rates of 80 common genes showed a slow evolutionary pace and low nucleotide variations, all genes being subjected to purifying selection.

Total genomic DNA was isolated from fresh leaves of a single individual using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA), and was used to prepare the shotgun library following the manufacturer’s protocol for Hiseq 4000 Sequencing System (Illumina, CA, USA). The library was sequenced by Nanjing Genepioneer Biotechnologies Inc. (Nanjing, China). Raw reads were obtained and trimmed using CLC Genomics Workbench v9 (CLC Bio, Aarhus, Denmark) with default parameters. The resultant clean reads were then employed to assemble the plastome using the program NOVOPlasty (Dierckxsens et al., 2017) with O. fragrans (GenBank: MG820121) (Duan et al., 2019) as the reference. The resultant genome was annotated by PGA (Qu et al., 2019). The plastomes generated in the present study are available in the NCBI Genbank database.