The genera Arnebia and Lithospermum (Lithospermeae-Boraginaceae) comprise 25–30 and 50–60 species, respectively. Some of them are economically valuable, as their roots frequently contain a purple-red dye used in the cosmetic industry. Furthermore, dried roots of Arnebia euchroma, A. guttata, and Lithospermum erythrorhizon, which have been designated Lithospermi Radix, are used as traditional Korean herbal medicine. This study is the first report on the floral micromorphology and complete chloroplast (cp) genome sequences of A. guttata (including A. tibetana), A. euchroma, and L. erythrorhizon. We reveal great diversity in floral epidermal cell patterns, gynoecium, and structure of trichomes. The cp genomes were 149,361–150,465 bp in length, with conserved quadripartite structures. In total, 112 genes were identified, including 78 protein-coding regions, 30 tRNA genes, and four rRNA genes. Gene order, content, and orientation were highly conserved and were consistent with the general structure of angiosperm cp genomes. Comparison of the four cp genomes revealed locally divergent regions, mainly within intergenic spacer regions (atpH-atpI, petN-psbM, rbcL-psaI, ycf4-cemA, ndhF-rpl32, and ndhC-trnV-UAC). To facilitate species identification, we developed molecular markers psaA- ycf3 (PSY), trnI-CAU- ycf2 (TCY), and ndhC-trnV-UAC (NCTV) based on divergence hotspots. High-resolution phylogenetic analysis revealed clear clustering and a close relationship of Arnebia to its Lithospermum sister group, which was supported by strong bootstrap values and posterior probabilities. Overall, gynoecium characteristics and genetic distance of cp genomes suggest that A. tibetana, might be recognized as an independent species rather than a synonym of A. guttata. The present morphological and cp genomic results provide useful information for future studies, such as taxonomic, phylogenetic, and evolutionary analysis of Boraginaceae.

For detailed floral micromorphological observations, fully mature reproductive organs were examined using a stereo microscope (SZX16; Olympus, Tokyo, Japan). For scanning electron microscopic observations, the dried samples from voucher specimens were rehydrated overnight in a wetting agent (Agepon: distilled water, 1:200) (Agfa Gevaert, Leverkusen, Germany). The floral samples were dehydrated through a graded ethanol series (50%, 70%, 90%, 95%, and 100% ethanol) at room temperature for 1 h at each ethanol concentration. The dehydrated material was immersed in liquid CO₂ for critical-point drying (SPI-13200JE-AB; SPI Supplies, West Chester, PA, USA) and subsequent mounting on aluminum stubs using a double-sided adhesive conductive carbon disk (05073-BA; SPI Supplies). All samples were coated with gold using an ion-sputtering device (208HR; Cressington Scientific Instruments Ltd., Watford, UK) and were observed using a low-voltage field emission scanning electron microscope (JSM-7600F; JEOL, Tokyo, Japan) at an accelerating voltage of 5–10 kV and working distance of 8–10 mm.