DOI: 10.5061/dryad.gtht76hk7
title: Dissection for floral micromorphology and plastid genome of valuable medicinal borages Arnebia and Lithospermum (Boraginaceae)

description: 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.
description: 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 CO<sub>2</sub> 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.