Phylogeny and historical biogeography of Lithospermeae (Boraginaceae): Disentangling the possible causes of Miocene diversifications.

Studies about the drivers of angiosperm clade diversifications have revealed how the environment continuously alters the species chances to adapt or to go extinct. This process depends on complex interactions between abiotic and biotic factors, conditioned to the geological and tectonic settings, the genetic variability of species and the rate at which speciation occurs. In this study, we aim to elucidate the timing of diversification of the Lithospermeae, the second largest tribe within Boraginaceae, and to identify the possible morphological and ecological characters associated with shifts in diversification rates of the most species-rich clades. Lithospermeae includes ca. 470 species and 26 genera, among which are some of the largest genera of the family such as Onosma (150 spp.), Echium (60 spp.), and Lithospermum (80 spp.). An exhaustive study of the whole clade is not available to date and its evolutionary history and diversification rates are incompletely known. In the present study, we provide the most comprehensive phylogeny of the group so far, sampling 242 species and all 26 genera. We found that crown-groups and diversification rates of Lithospermeae largely date back to the Mid-Miocene, with high diversification rates in the largest genera, though only significantly high in Onosma. Our analysis fails to associate any of the functional or morphological traits considered with significant shifts in diversification rates. The timing of the diversification of the species-rich clades corresponds with Miocene tectonic events and global climate changes increasing aridity across Eurasia and western North America. These results suggest a causal link between known ecological features of Lithospermeae (i.e., pre-adaptation to arid, open habitats, and mineral soils) and their diversification. Future studies should expand the sampling of individual subclades and detailed functional analyses to identify the contribution of adaptations to arid conditions and pollinator shifts.

Associations between sex-organ deployment and morph bias in related heterostylous taxa with different stylar polymorphisms.

PREMISE OF THE STUDY: Populations of heterostylous species are characterized by two or three floral morphs with reciprocal positioning of stigmas and anthers. Theoretical models predict equal morph frequencies (isoplethy) when disassortative mating is prevalent in populations, but biased morph ratios may occur when variation in the expression of heterostyly causes deviations from intermorph mating. METHODS: We explore the role of sex-organ deployment in governing morph ratios in two closely related genera of Boraginaceae, exhibiting striking variation in floral traits associated with the heterostylous syndrome. We sampled 66 populations of six species of Glandora and 39 populations of three species of Lithodora across their distributional range in the Mediterranean. In each population we estimated morph ratios and measured several floral traits. We used phylogenetically corrected and noncorrected regressions to test the hypothesis that differences in sex-organ reciprocity and herkogamy are associated with deviations from isoplethy. KEY RESULTS: Biased morph ratios occurred in 24% of populations, particularly in Lithodora. Populations biased for the long-styled morph (L-morph) were more frequent than the short-styled morph (S-morph). Distylous species were less likely to exhibit biased ratios than species with stigma-height dimorphism. In Lithodora fruticosa, a species lacking reciprocity, decreased herkogamy in the S-morph was associated with increasing L-morph bias, perhaps resulting from self-interference. CONCLUSION: Striking variation in the expression of heterostyly in Glandora and Lithodora is associated with biased morph ratios, which probably result from pollinator-mediated mating asymmetries within populations.

Survey of pyrrolizidine alkaloids in seven varieties of Lappula squarrosa: An alternative source of heart-healthy vegetable oil.

INTRODUCTION: Growing demand for heart-healthy omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is putting stress on wild fish stocks. There is now a compelling need for new and novel sources of non-traditional seed oils containing high stearidonic acid (SDA), a precursor of EPA and DHA, to reduce this demand. The seed oil of Lappula squarrosa is one of the richest sources of SDA, however, the plant has been found to contain toxic pyrrolizidine alkaloids (PAs). OBJECTIVE: In this study, the PA concentrations of seven varieties (A-G) of Lappula squarrosa were analysed to determine the most suitable varieties for commercial seed oil production. METHODS: Whilst the clean-up procedure for the PAs in the roots, flowers and leaves was on diatomaceous earth columns and finally analysed with GC-EI-MS, that of the seeds was through SCX-SPE and a more sensitive HPLC-ESI-MS/MS sum parameter method was used in the analysis. RESULTS: Altogether six PAs (supinine, amabiline, intermedine, lycopsamine and 3'-acetylintermedine) including one unknown retronecine-type PA were identified with variety C recording the lowest total PA concentration (4.64 mg seneciphylline equivalents (SE)/g dry weight (d.w.)). Besides, the total PA concentrations in the seeds of Lappula squarrosa varieties ranged between 2.88 µg PA/g and 10.36 µg PA/g d.w. CONCLUSION: Based solely on overall PA concentrations and PA distribution, variety D (5.95 mg SE/g d.w.) was found to be a potential candidate for commercial seed oil cultivation.

Fossil nutlets of Boraginaceae from the continental Eocene of Hamada of Méridja (southwestern Algeria): The first fossil of the Borage family in Africa.

PREMISE OF THE STUDY: The Paleogene deposits of the Hamada of Méridja, southwestern Algeria, are currently dated as lower-to-middle Eocene in age based on fossil gastropods and charophytes. Here we report the presence of fruits that can be assigned to the Boraginaceae s.str., apparently representing the first fossil record for this family in Africa, shedding new light on the historical biogeography of this group. METHODS: Microscopic studies of the fossil nutlets were carried out and compared to extant Boraginaceae nutlets, and to types reported in the literature for this family. KEY RESULTS: The fossils are strikingly similar in general size and morphology, particularly in the finer details of the attachment scar and ornamentation, to nutlets of extant representatives of the Boraginaceae tribe Echiochileae, and especially the genus Ogastemma. We believe that these nutlets represent an extinct member of this lineage. CONCLUSIONS: The Ogastemma-like fossils indicate that the Echiochileae, which are most diverse in northern Africa and southwestern Asia, have a long history in this region, dating back to the Eocene. This tribe corresponds to the basal-most clade in Boraginaceae s.str., and the fossils described here agree well with an assumed African origin of the family and the Boraginales I, providing an important additional calibration point for dating the phylogenies of this clade.

[In silico cloning and bioinformatics analysis of an AP2/EFR family gene from Arnebia euchroma].

A cDNA sequence of Arnebia euchroma AP2/ERF named AeAP2/ERF was cloned by in silico cloning in this study, using ACX71873 sequence from Lithospermum erythrorhizon as the probe sequence. Some characters of the AP2/ERF gene and encoded protein sequences were predicted and analyzed by the bioinformatics methods, including general physical and chemical properties, hydrophobieity, signal peptide, secondary structure, localization sites in cells. Results showed that the 876 bp long gene included a 1 077 bp ORF and encoding 205 amino acid. The AeAP2/ERF protein had no signal peptide, it was a hydrophilic proteins located in nucleus. The function of the AP2/ERF protein was mainly involved with metabolism controlling and signal transduction.

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.

Unusual heterostyly: style dimorphism and self-incompatibility are not tightly associated in Lithodora and Glandora (Boraginaceae).

BACKGROUND AND AIMS: Heterostyly is a floral polymorphism characterized by the reciprocal position of stamens and stigmas in different flower morphs in a population. This reciprocal herkogamy is usually associated with an incompatibility system that prevents selfing and intra-morph fertilization, termed a heteromorphic incompatibility system. In different evolutionary models explaining heterostyly, it has been alternately argued that heteromorphic incompatibility either preceded or followed the evolution of reciprocal herkogamy. In some models, reciprocal herkogamy and incompatibility have been hypothesized to be linked together during the evolution of the heterostylous system. METHODS: We examine the incompatibility systems in species with different stylar polymorphisms from the genera Lithodora and Glandora (Boraginaceae). We then test whether evolution towards reciprocal herkogamy is associated with the acquisition of incompatibility. To this end, a phylogeny of these genera and related species is reconstructed and the morphological and reproductive changes that occurred during the course of evolution are assessed. KEY RESULTS: Both self-compatibility and self-incompatibility are found within the studied genera, along with different degrees of intra-morph compatibility. We report for the first time extensive variability among members of the genus Glandora and related species in terms of the presence or absence of intraspecies polymorphism and heteromorphic incompatibility. Overall, our results do not support a tight link between floral polymorphism and incompatibility systems. CONCLUSIONS: The independent evolution of stylar polymorphism and incompatibility appears to have occurred in this group of plants. This refutes the canonical view that there is strong linkage between these reproductive traits.

Pyrrolizidine alkaloid-containing toxic plants (Senecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium, and Echium spp.).

Pyrrolizidine alkaloid (PA)-containing plants are found throughout the world and are probably the most common plant cause of poisoning of livestock, wildlife, and humans. PAs are potent liver toxins that under some conditions can be carcinogenic. This article briefly introduces high-risk North American PA-containing plants, summarizing their toxicity and subsequent pathology. Current diagnostic techniques, treatments, and strategies to avoid losses to PA poisoning are also reviewed.

Comparative study on enantiomeric excess of main akannin/shikonin derivatives isolated from the roots of three endemic Boraginaceae plants in China.

This work systematically investigated the enantiomeric excess (e.e.) of main components isolated from the roots of three endemic Boraginaceae plants distributed extensively in China, named Arnebia euchroma (Royle) Johnst (A.e.), Lithospermum erythrorhizon Sieb. et Zucc. (L.e.) and Onosma confertum W. W. Smith (O.c.), and the optical purity of their hydrolysis products separately, by means of three different approaches. The influence of HCl on the e.e. values of the major constituents was also studied. Analysis of the absolute configurations and e.e. values of all the derivatives acquired was performed by CD and chiral-HPLC respectively. The results of the main constituents demonstrated that A.e. mainly yields S-form naphthoquinone derivatives, while the R-form is predominant in the derivatives of L.e. and O.c. The optical purity of alkannin and shikonin and their derivatives was not influenced by acid treatment in the course of separation and hydrolysis. Additionally, it was found that 100% e.e. of shikinon could be acquired from a specific shikinon ester derivative, ß,ß-dimethylacrylshikonin occurring in the roots of O.c., as did 100% e.e. of alkannin from ß,ß-dimethylacrylalkannin contained in the roots of A.e.

Origin and evolution of endemic Galápagos Varronia species (Cordiaceae).

Four endemic Varronia species (Cordiaceae) occupy the Galápagos archipelago. Three comprise the V. leucophlyctis complex (V. anderssonii, V. leucophlyctis, V. scouleri), whose species' limits are not well defined but that is morphologically distinct from the fourth endemic species, V. revoluta. Sequence data from the nuclear rDNA ITS region and the cpDNA ndhF gene were gathered from 49 accessions of Varronia from five Galápagos islands in order to test the evolutionary relationships of endemic Varronia species, determine the number of immigration events to the islands and estimate their age of origin. All endemic species nest within the clade of species belonging to Varronia, which is an entirely American genus. We find little evidence of phylogenetic structuring of the V. leucophlyctis complex but divergent phylogenetic signals from nuclear and chloroplast genomes regarding its relationship to V. revoluta. Results are consistent with a hybridization event involving ancestral Galapagean lineages, with chloroplast and nuclear data suggesting one or two dispersal events from the Americas to the Galápagos, respectively. Fossil-based divergence time estimates indicate endemic species diverged from American continental species as early as 4.5 Myr ago and radiated 1.12 Myr, which coincides with ages of exposed and subsided Galápagos islands.

Differentiation of Symphytum species using RAPD and seed fatty acid patterns.

RAPD and fatty acid profiles, and alpha-tocoferol contents of the seeds of some Symphytum species were analyzed for their differentiation. Discriminative patterns were found in the examined species. Genotypic characteristics of three Symphytum species were experienced using 20 decamer RAPD primers. The majority of band positions varied between species. The total amplified products of 20 RAPD primers was 247 (average of 12.35 bands per primer), of which 189 bands were polymorphic, corresponding to nearly 76.1% genetic diversity. The number of bands for each RAPD primer varied from 8 (UBC320) to 18 (OPA7). The percent of polymorphic bands ranged from 45.4% (OPJ20) to 93.3% (OPA16). Major unsaturated fatty acids in the seeds were linoleic, gamma-linolenic and oleic acids; alpha-linolenic and eicosenoic acids exhibited lower levels. Variations in quantities and total ratios of fatty acid groups, and alpha-tocopherol contents were also observed between the species, and the developmental stages of the seeds. Significant differences were found for the whole series of fatty acids between species, in addition to the calculated ratios (p < 0.05). Differences were also significant for fatty acid traits between two developmental stages of the seeds of endemic S. pseudobulbosum (p < 0.05). In some morphological descriptors of the seeds, difference was found at a significant level (p < 0.05). Obtained data based on genetic and biochemical variations seem to be useful for molecular delimitation of Symphytum, in addition to selection of the genotypes expressing a high amount of GLA.

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.

Flavonoids from Ficaria verna Huds.

A phytochemical investigation of the flowers and leaves of Ficaria verna Huds. (Ranunculaceae) yielded four additional known flavonoid compounds including: kaempferol 3-O-beta-D-(6"-a-L-rhamnopyranosyl)-glucopyranoside (nicotiflorin), apigenin 8-C-beta-D-glucopyranoside (vitexin), luteolin 8-C-beta-D-glucopyranoside (orientin) and apigenin 8-C-beta-D-(2"-O-beta-D-glucopyranosyl)-glucopyranoside (flavosativaside). The characterisation of these compounds was achieved by various chromatographic and spectroscopic methods (UV, 1H NMR, 13C NMR and MS).

On the occurrence of exudate flavonoids in the borage family (Boraginaceae).

Externally accumulated flavonoid aglycones have been found for the first time in Nonea species. They exhibit only flavones, one of them being the rare tricetin-4'-methyl ether. Within the subfamily Boraginoideae, exudate flavonoids appear to be a rare character.

Occurrence and characterization of oils rich in gamma-linolenic acid (III): the taxonomical value of the fatty acids in Echium (Boraginaceae).

Fourteen species of the genus Echium (Fam. Boraginaceae) collected in the Macaronesia were surveyed in a search for high levels of gamma-linolenic acid (GLA, 18:3omega6) in the seed oil. High amounts of this fatty acid were found in all of them, ranging from 18.85% (E. pitardii var. pitardii) to 27.42% (E. gentianoides) on total seed fatty acids. The GLA content related to total seed weight was also significant, ranging from 1.26% (E. handiense) to 8.22% (E. gentianoides). In addition, considerable amounts of stearidonic acid (SA, 18:4omega3) were detected, ranging from 3.78% (E. bonnetii var. bonnetii) to 8.81% (E. pininana) on total fatty acids. Besides all the perennial species, the four herbaceous Echium taxa endemic to the Macaronesia also showed high GLA percentages. This is in contrast to the low GLA level found in continental Echium species, all of them bearing an herbaceous habit. These results are in good agreement with the available genetic data and show the ability of GLA to discriminate between Macaronesian and continental Echium species. The analysis of five other Macaronesian species belonging to plant families rich in GLA are also reported.