Abstract— Utilizing 10 cpDNA regions and thorough taxon sampling, a phylogeny is reconstructed for Lithospermum and related members of both Lithospermeae, the tribe to which it is assigned, and Boraginaceae. Lithospermum is supported as monophyletic, and
the genus is hypothesized to have originated in the Old World, after which there was one colonization of the New World. The heterostylous breeding system is inferred to have originated within Lithospermum either seven times or six times with one loss, and with other independent origins
within Lithospermeae. The stability of the 10-region matrix is investigated, as are the number and combination of regions necessary to accurately reconstruct phylogenies. The combination of concatenated regions is important, and the following regions are recommended for future phylogenetic
studies of genera of Boraginaceae: the rpl16 intron, matK, psbA-trnH, trnL-rpl32, and trnQ-rps16. The use of these recommended regions is conservative, and it contrasts with most intrageneric studies of Boraginaceae, which often are based
on the trnL-trnF spacer and nuclear ribosomal ITS.