Comparative Analysis of Shikonin and Alkannin Acyltransferases Reveals Their Functional Conservation in Boraginaceae.

Shikonin and its enantiomer, alkannin, are bioactive naphthoquinones produced in several plants of the family Boraginaceae. The structures of these acylated derivatives, which have various short-chain acyl moieties, differ among plant species. The acylation of shikonin and alkannin in Lithospermum erythrorhizon was previously reported to be catalyzed by two enantioselective BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1). However, the mechanisms by which various shikonin and alkannin derivatives are produced in Boraginaceae plants remain to be determined. In the present study, evaluation of six Boraginaceae plants identified 23 homologs of LeSAT1 and LeAAT1, with 15 of these enzymes found to catalyze the acylation of shikonin or alkannin, utilizing acetyl-CoA, isobutyryl-CoA or isovaleryl-CoA as an acyl donor. Analyses of substrate specificities of these enzymes for both acyl donors and acyl acceptors and determination of their subcellular localization using Nicotiana benthamiana revealed a distinct functional differentiation of BAHD acyltransferases in Boraginaceae plants. Gene expression of these acyltransferases correlated with the enantiomeric ratio of produced shikonin/alkannin derivatives in L. erythrorhizon and Echium plantagineum. These enzymes showed conserved substrate specificities for acyl donors among plant species, indicating that the diversity in acyl moieties of shikonin/alkannin derivatives involved factors other than the differentiation of acyltransferases. These findings provide insight into the chemical diversification and evolutionary processes of shikonin/alkannin derivatives.

[Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells].

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 µmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/ß-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, ß-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3ß, ß-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3ß protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, ß-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/ß-catenin signaling pathway.

Development of the first microsatellite markers using high-throughput sequencing for a hexaploid coastal species, Tournefortia argentea L. f. (Boraginaceae).

BACKGROUD: Tournefortia argentea L. f. is a hexaploid shrub or tree species with ecological and evolutionary significances, which forms the fringe of vegetation closest to the sea on tropical coral islands. Previous studies have never addressed on genetic information, and thus genomic resources remain scarce. METHODS AND RESULTS: We used nine individuals from different islands to identify polymorphic microsatellites of T. argentea by Illumina high-throughput sequencing. Thirty-five polymorphic microsatellite markers were developed. Characteristics of each locus were tested using 48 individuals collected from three populations of T. argentea. A total of 320 alleles were found across the 35 microsatellite loci. The number of alleles per locus ranged from 5 to 15, with an average of 9.1. Observed and expected heterozygosities in each locus per population varied from 0.000 to 1.000 and from 0.000 to 0.893, respectively. CONCLUSIONS: In this study, we report the development of 35 polymorphic microsatellite markers based on Illumina high-throughput sequencing. These markers will facilitate the investigations of genetic diversity, population structures and evolutionary history of T. argentea.

[Cloning and functional verification of carboxyl CoA ligases(AeCCLs) in Arnebia euchroma].

Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.

[Cloning and functional analysis of caffeic acid and rosmarinic acid glycosyltransferases from Arnebia euchroma].

Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.

Persistence of Pseudomonas fluorescens LBUM677 in the rhizosphere of corn gromwell (Buglossoides arvensis) under field conditions and its impact on seed oil and stearidonic acid bioaccumulation.

AIMS: The aim of this study was to evaluate the persistence of Pseudomonas fluorescens LBUM677 in the rhizosphere of Buglossoides arvensis under agricultural field conditions and determine if B. arvensis intraspecific genetic variations affect the capacity of LBUM677 to colonize its rhizosphere and increase seed oil and stearidonic acid (SDA) accumulation. METHODS AND RESULTS: Two field experiments were performed to: (i) study the persistence of various concentrations of LBUM677 inoculated in the rhizosphere of B. arvensis and determine a minimum inoculation threshold required to maximize biological activity; and (ii) study the impact of B. arvensis intraspecific genetic variations on LBUM677 rhizosphere colonization and seed oil and SDA accumulation. In order to track LBUM677 populations in soil over time, a specific quantitative polymerase chain reaction assay was developed. Inoculation with a minimum of 109 LBUM677 bacterial cells per plant was determined as a threshold to promote maximum B. arvensis rhizosphere colonization and seed oil and SDA accumulation. Buglossoides arvensis intraspecific genetic variations had an impact on rhizosphere colonization, B. arvensis seed oil and SDA accumulation, where two cultivars benefited more than others from LBUM677 inoculation. CONCLUSIONS: LBUM677 can colonize the rhizosphere and increase seed oil and SDA yields in B. arvensis plants in a cultivar-dependant manner. SIGNIFICANCE AND IMPACT OF THE STUDY: LBUM677 shows potential to be used as a biofertilizer to specifically increase seed oil and SDA yields in B. arvensis. This will in turn promote the development of an economically viable agricultural-based approach as an alternative for producing high-quality polyunsaturated fatty acids.

Flow cytometric seed screen data are consistent with models of chromosome inheritance in asymmetrically compensating allopolyploids.

Angiosperms have evolved a mechanism of double fertilization, which results in the production of a separate embryo (new individual) and endosperm (nutritive tissue). The flow cytometric seed screen (FCSS) was developed to infer plant reproduction modes based on endosperm-to-embryo DNA content ratio (Pind ). A ratio of 1.5 indicates sexual reproduction, whereas higher values of ≥2.0 are consistent with apomixis. Although FCSS has been successfully applied to the study of sexual and asexual plants, the limits of FCSS and particularly its potential for determination of reproduction modes in hemisexual plants have not been explored. Here, we evaluated the application of FCSS to the study of reproduction modes in two asymmetrically compensating allopolyploids (ACAs), Onosma arenaria and Rosa canina. These two species are characterized by the presence of asexually inherited univalent-forming and sexually inherited bivalent-forming chromosome sets. They both use asymmetric meiosis, which eliminates univalent-forming chromosome sets from the male gamete and retains them in the female gamete. Different chromosomal behavior in male and female meiosis in these plants is reflected in different theoretically derived Pind values, which deviate from a sexual 1.5 value. Here, we determined Pind FCSS-based values in seeds of ACAs, and compared the results to sexual species. As expected, we determined that the mean Pind is 1.51, 1.52, and 1.52 in the sexual plants, that is, Capsella bursa-pastoris, Crataegus monogyna, and O. pseudoarenaria, respectively. In the ACAs, different mean Pind values were determined for O. arenaria (1.61) and R. canina (1.82). These values are consistent with the theoretical Pind values determined based on models of chromosome inheritance. This study highlights the precision of flow cytometry in determining DNA content and it's utility in screening reproduction modes. Additionally, it advocates for more in-depth investigations into rapid screening of accessions where the Pind ratio has deviated from the 1.5 value typical of sexual species, which may indicate meiotic irregularities.

Reproductive Strategy of the Polyploid Species Varronia curassavica Jacq. in Restinga Environment.

This study aimed to elucidate the breeding strategies of Varronia curassavica, an important medicinal species associated with Brazilian restinga. This was accomplished by combining phenological and genetic data. Every 2 weeks over a period of 2 years, we measured flowering and fruiting phenology to evaluate the activity and intensity of phenophases (n = 60). We evaluated the mating system, pollen ovule ratio and genotypes from progeny and mother plants using 8 nuclear microsatellite loci. We observed flowering and fruiting of V. curassavica at low intensity throughout the entire year, but with 2 distinct peaks, one of which was seasonal, corresponding to the period of gradual increase of temperature and photoperiod. Overlapping of flowering and fruiting strategies favors gene flow among different groups of individuals and between populations by attraction of fauna throughout the year. Analysis of the mating system indicates that V. curassavica is a typical outcrossed species (t^ = 0.98; pollen/ovule ratio = 7087.50). Combining phenology with genetic studies improved our understanding of the reproductive strategies of this species. The typical outcrossing system of V. curassavica reflects the existence of functional self-incompatibility mechanisms still unaffected by changes in genetic balance by polyploidy.

[Transcriptome-based gene mining and bioinformatics analysis of p-hydroxybenzoate geranyltransferase genes in Arnebia euchroma].

The p-hydroxybenzoate geranyltransferases(PGT) play an important role in the biosynthesis pathways of shikonin derivatives. Six PGTs were obtained from transcriptome datebase of Arnebia euchroma by using bioinformatics methods and the proteins＇physiochemical properties they encoded were predicted. The result of protein domain prediction showed all of the six protein sequences contained the conserved domain of Ubia prenyltransferase family and possessed the motif NDxxDxxxD for prenyldiphosphate binding and a GX(K/Y)STAL sequence for putative aromatic ring binding. The phylogenetic tree showed that PGT and p-hydroxybenzoate polyprenyltransferase(PPT) belonged to two different clades. The results of gene expression analyses showed that the expression levels in the red shikonin-proficient line and the overground part of A. euchroma that could produce shikonin derivatives was much higher than the white shikonin-deficient line and the underground part, which suggested a positive correlation between the expression levels of PGT genes and shikonin production. This study aims to lay a foudation for further understanding of the function and enzymatic properties of PGT and provide a basis for the biosynthesis pathways and metabolic regulation of shikonin derivatives.

Cytomictic anomalous male meiosis and 2n pollen grain formation in Mertensia echioides Benth. (Boraginaceae) from Kashmir Himalaya.

Presently Mertensia echioides Benth. (Boraginaceae) collected from Kashmir Himalaya, India, is cytologically analyzed for the first time revealing 2n = 2x = 24 (diploid). Interestingly we found 4.3-6.2% syncytic meiocytes/PMCs with 2n = 4x = 48 (tetraploid) in addition to normal meiocytes (2n = 24) during male meiosis. These comparatively larger PMCs (pollen mother cells) lead to the formation of fertile giant 2n pollen grains. A frequency of 6.4-13.3% PMCs shows transfer of chromatin material at prophase-I and, therefore, results in aneuploid meiocytes. Whole chromatin transfer by the process of cytomixis could also have led to the formation of tetraploid cells. Translocation heterozygosity is also evident in the form of multivalents in 12-17% diploid (2x) meiocytes at diakinesis and metaphase-I and is reported for the first time in this species. The syncytes formed depict open chain hexavalent and quadrivalent formation in the three populations with different frequencies. Moreover chromatin stickiness at metaphase-I is observed in 45% of PMCs in population-1 (P-1). Syncyte or unreduced PMC formation leading to unreduced fertile gametes is here speculated to act as a possible way out for infraspecific polyploidization in the species.

Effects of exogenous methyl jasmonate on the biosynthesis of shikonin derivatives in callus tissues of Arnebia euchroma.

The shikonin derivatives, accumulated in the roots of Arnebia euchroma (Boraginaceae), showed antibacterial, anti-inflammatory, and anti-tumor activities. To explore their possible biosynthesis regulation mechanism, this paper investigated the effects of exogenous methyl jasmonate (MJ) on the biosynthesis of shikonin derivatives in callus cultures of A. euchroma. The main results include: Under MJ treatment, the growth of A. euchroma callus cultures was not inhibited, but the expression level of both the genes involved in the biosynthesis of shikonin derivatives and their precursors and the genes responsible for intracellular localization of shikonin derivatives increased significantly in the Red Strain (shikonin derivatives high-producing strain). The quantitative analysis showed that six out of the seven naphthoquinone compounds under investigation increased their contents in the MJ-treated Red Strain, and in particular, the bioactive component acetylshikonin nearly doubled its content in the MJ-treated Red Strain. In addition, it was also observed that the metabolic profiling of naphthoquinone compounds changed significantly after MJ treatment, and the MJ-treated and MJ-untreated strains clearly formed distinct clusters in the score plot of PLS-DA. Our results provide some new insights into the regulation mechanism of the biosynthesis of shikonin derivatives and a possible way to increase the production of naphthoquinone compounds in A. euchroma callus cultures in the future.

Identification and functional analysis of a Δ6-desaturase gene and the effects of temperature and wounding stresses on its expression in Microula sikkimensis leaves.

A Δ6-desaturase gene was isolated from Microula sikkimensis. Sequence analysis indicated that the gene, designated MsD6DES, had an open reading frame of 1,357 bp and encoded 448 amino acids. Heterologous expression in tobacco indicated that MsD6DES can use endogenous substrates to synthesize γ-linolenic acid (GLA, 18:3(Δ 6,9,12)) and octadecatetraenoic acid (OTA, 18:4(Δ 6,9,12,15)). MsD6DES transcripts were distributed in all tested tissues, with high expression levels in seeds and young leaves. The effects of temperature and wounding stresses on MsD6DES expression were analyzed. The results indicated that temperature regulates MsD6DES at the transcriptional level. MsD6DES expression increased first, reaching a maximum 4 h after low-temperature treatment. A slight increase in MsD6DES transcript levels was also observed under high temperature. We found that the response of MsD6DES to temperature stress was different from those of fungi and algae. In addition, MsD6DES was found to be wound-inducible.

Multiple origins for Hound's tongues (Cynoglossum L.) and Navel seeds (Omphalodes Mill.)--the phylogeny of the borage family (Boraginaceae s.str.).

Recent studies all indicated that both the affinities and subdivision of Boraginaceae s.str. are unsatisfactorily resolved. Major open issues are the placement and affinities of Boraginaceae s.str. in Boraginales and the major clades of the family, with especially the large tribes Cynoglosseae and Eritrichieae repeatedly retrieved as non-monophyletic groups, and the doubtful monophyly of several larger genera, especially Cynoglossum and Omphalodes. The present study addresses and solves these questions using two plastid markers (trnL-trnF, rps16) on the basis of a sampling including 16 outgroup taxa and 172 ingroup species from 65 genera. The phylogeny shows high statistical support for most nodes on the backbone and on the individual clades. Boraginaceae s.str. are sister to African Wellstediaceae, Wellstediaceae-Boraginaceae s.str. is sister to African Codonaceae. Echiochileae are retrieved as sister to the remainder of Boraginaceae s.str., which, in turn, fall into two major clades, the Boragineae-Lithospermeae (in a well-supported sister relationship) and the Cynoglosseae s.l. (including Eritrichieae). Cynoglosseae s.l. is highly resolved, with Trichodesmeae (incl. Microcaryum, Lasiocaryum) as sister to the remainder of the group. Eritrichieae s.str. (Eritrichium, Hackelia, Lappula) are resolved on a poorly supported polytomy together with the Omphalodes-clade (incl. Myosotidium, Cynoglossum p.p.), and the Mertensia-clade (incl. O. scorpioides, Asperugo). The Myosotideae (Myosotis, Trigonotis, Pseudomertensia) are retrieved in a well-supported sister-relationship to the core-Cynoglosseae, the latter comprising all other genera sampled. Cynoglossum is retrieved as highly para- and polyphyletic, with a large range of generic segregates embedded in Cynoglossum, but other species of Cynoglossum are sister to Microula or to the American "Eritrichieae" (Cryptantha and allied genera). Representatives of the genus Cynoglossum in its current definition are segregated onto six independent lineages, members of Omphalodes onto three independent lineages. At least 11 of the genera here sampled are deeply nested in other genera. The data show that individual details of nutlet morphology (e.g., winged margins, glochidia) are highly homoplasious. Conversely, a complex of nutlet characters (e.g., characters of the gynobase and cicatrix together with nutlet orientation and sculpturing) tends to circumscribe natural units. Geographical distribution of major clades suggests that the family originated in Africa and western Asia and radiated to eastern Eurasia, with several independent dispersal events into Australia and the New World.

Specific genes of cytochrome P450 monooxygenases are implicated in biosynthesis of caffeic acid metabolites in rolC-transgenic culture of Eritrichium sericeum.

Expression of rol agrobacterial oncogenes in plant cell cultures is known to result in activation of secondary metabolite biosynthesis. In the present work, we show that rolC can activate expression of key genes of secondary metabolism using the rolC-transgenic culture of Eritrichium sericeum producing caffeic acid metabolites (rosmarinic acid and rabdosiin) as an example. Increased content of rosmarinic acid in the rolC-transformed callus culture resulted from transcriptional activation of members of the CYP98 family of plant cytochrome P450-containing monooxygenase genes. The rolC gene expression led to increased transcript abundance of the CYP98A3 subfamily members, which are closely related homologs of CYP98A6 of Lithospermum erythrorhizon and are known to be key genes in rosmarinic acid biosynthesis. In contrast, expression of other CYP genes, such as CYP98A1 and CYP98A2, which are not implicated in rosmarinic acid biosynthesis, was not activated in the rolC-transformed calluses. These results are indicative of selective effect of rolC on transcription of particular genes implicated in secondary metabolism.

Molecular systematics of Boraginaceae tribe Boragineae based on ITS1 and trnL sequences, with special reference to Anchusa s.l.

BACKGROUND AND AIMS: Boragineae is one of the main tribes of Boraginaceae, but delimitation and intergeneric classification of this group are unclear and have not yet been studied using DNA sequences. In particular, phylogenetic relationships in Anchusa s.l. still need to be elucidated in order to assess its taxonomic boundaries with respect to the controversial segregate genera Hormuzakia, Gastrocotyle, Phyllocara and Cynoglottis. METHODS: Phylogenetic relationships among 51 taxa of tribe Boragineae were investigated by comparative sequencing of the trnL(UAA) intron of the plastid genome and of the ITS1 region of the nuclear ribosomal DNA. Exemplar taxa from 16 genera of Boragineae and all subgenera of Anchusa s.l. were included, along with two selected outgroups from tribes Lithospermeae and Cynoglosseae. KEY RESULTS: Phylogenies generated by maximum parsimony and combined ITS1-trnL sequences support the monophyly of the tribe and a split into two clades, Pentaglottis and the remainder of Boragineae. The latter contains two large monophyletic groups. The first consists of three moderately to well-supported branches, Borago-Symphytum, Pulmonaria-Nonea and Brunnera. In the Pulmonaria-Nonea subclade, the rare endemic Paraskevia cesatiana is sister to Pulmonaria, and Nonea appears to be paraphyletic with respect to Elizaldia. The second main group corresponds to the well-supported clade of Anchusa s.l., with the megaphyllic, polyploid herb Trachystemon orientalis as sister taxon, although with low support. Anchusa s.l. is highly paraphyletic to its segregate genera and falls into four subclades: (1) Phyllocara, Hormuzakia, Anchusa subgenus Buglossum and A. subgenus Buglossoides; (2) Gastrocotyle; (3) A. subgenus Buglossellum and Cynoglottis; and (4) A. subgenus Anchusa, Lycopsis and Anchusella. All species of Anchusa subg. Anchusa, including the South African A. capensis, are included in a single unresolved clade. Anchusa subgenus Limbata is also included here despite marked divergence in floral morphology. The low nucleotide variation of ITS1 suggests a recent partly adaptive radiation within this group. CONCLUSIONS: Molecular data show that nine of the usually accepted genera of the Boragineae consisting of two or more species are monophyletic: Anchusella, Borago, Brunnera, Cynoglottis, Gastrocotyle, Hormuzakia, Nonea, Pulmonaria and Symphytum. In addition, the tribe includes the four monotypic genera Paraskevia, Pentaglottis, Phyllocara and Trachystemon. The morphologically well-characterized segregate genera in Anchusa s.l. are all confirmed by DNA sequences and should be definitively accepted. Most of the traditionally recognized subgenera of Anchusa are also supported as monophyletic groups by both nuclear and plastid sequence data. In order to bring taxonomy in line with phylogeny, the institution of new, independent generic entities for subgenera Buglossum, Buglossellum and Buglossoides and a narrower but more natural concept of Anchusa are advocated.