Taxonomy, comparative genomics of Mullein (Verbascum, Scrophulariaceae), with implications for the evolution of Verbascum and Lamiales.

BACKGROUND: The genus Verbascum L. (Scrophulariaceae) is distributed in Africa, Europe, and parts of Asia, with the Mediterranean having the most species variety. Several researchers have already worked on the phylogenetic and taxonomic analysis of Verbascum by using ITS data and chloroplast genome fragments and have produced different conclusions. The taxonomy and phylogenetic relationships of this genus are unclear. RESULTS: The complete plastomes (cp) lengths for V. chaixii, V. songaricum, V. phoeniceum, V. blattaria, V. sinaiticum, V. thapsus, and V. brevipedicellatum ranged from 153,014 to 153,481 bp. The cp coded 114 unique genes comprising of 80 protein-coding genes, four ribosomal RNA (rRNA), and 30 tRNA genes. We detected variations in the repeat structures, gene expansion on the inverted repeat, and single copy (IR/SC) boundary regions. The substitution rate analysis indicated that some genes were under purifying selection pressure. Phylogenetic analysis supported the sister relationship of (Lentibulariaceae + Acanthaceae + Bignoniaceae + Verbenaceae + Pedaliaceae) and (Lamiaceae + Phyrymaceae + Orobanchaceae + Paulowniaceae + Mazaceae) in Lamiales. Within Scrophulariaceae, Verbascum was sister to Scrophularia, while Buddleja formed a monophyletic clade from (Scrophularia + Verbascum) with high bootstrap support values. The relationship of the nine species within Verbascum was highly supported. CONCLUSION: Based on the phylogenetic results, we proposed to reinstate the species status of V. brevipedicellatum (Engl.) Hub.-Mor. Additionally, three genera (Mazus, Lancea, and Dodartia) placed in the Phyrymaceae family formed a separate clade within Lamiaceae. The classification of the three genera was supported by previous studies. Thus, the current study also suggests the circumscription of these genera as documented previously to be reinstated. The divergence time of Lamiales was approximated to be 86.28 million years ago (Ma) (95% highest posterior density (HPD), 85.12-89.91 Ma). The complete plastomes sequence data of the Verbascum species will be important for understanding the Verbascum phylogenetic relationships and evolution in order Lamiales.

Molecular Characterization of Verbascum anisophyllum (Scrophulariaceae) Genetic Resources Through Inter-Simple Sequence Repeat (ISSR) Markers.

Verbascum (Mullein) flowers are highly valued as natural remedy for various respiratory diseases. Verbascum anisophyllum Murb. is a Balkan endemic, protected by law and included in the Bulgarian Red Data Book as "Critically Endangered". Thus, a strict conservation policy and a reliable evaluation of its genetic resources are required, considering its narrow distribution range and the increasing risk from destruction of its habitats. Here, we used Inter-simple sequence repeat (ISSR) markers to characterize the genetic diversity and to assess the genetic differentiation between the existing populations of VerbascUM anisophyllum in Bulgaria. The level of genetic diversity found herein clearly indicates a long-term potential for adaptability of this endangered plant. Our findings provide important knowledge of population genetic structure of this species, thus representing a strategy for its efficient conservation and utilization.

Genetic transformation of rare Verbascum eriophorum Godr. plants and metabolic alterations revealed by NMR-based metabolomics.

OBJECTIVES: To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. RESULTS: Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. CONCLUSION: Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

Combining optimal defense theory and the evolutionary dilemma model to refine predictions regarding plant invasion.

Optimal defense theory posits that plants with limited resources deploy chemical defenses based on the fitness value of different tissues and their probability of attack. However, what constitutes optimal defense depends on the identity of the herbivores involved in the interaction. Generalists, which are not tightly coevolved with their many host plants, are typically deterred by chemical defenses, while coevolved specialists are often attracted to these same chemicals. This imposes an "evolutionary dilemma" in which generalists and specialists exert opposing selection on plant investment in defense, thereby stabilizing defenses at intermediate levels. We used the natural shift in herbivore community composition that typifies many plant invasions to test a novel, combined prediction of optimal defense theory and the evolutionary dilemma model: that the within-plant distribution of defenses reflects both the value of different tissues (i.e., young vs. old leaves) and the relative importance of specialist and generalist herbivores in the community. Using populations of Verbascum thapsus exposed to ambient herbivory in its native range (where specialist and generalist chewing herbivores are prevalent) and its introduced range (where only generalist chewing herbivores are prevalent), we illustrate significant differences in the way iridoid glycosides are distributed among young and old leaves. Importantly, high-quality young leaves are 6.5x more highly defended than old leaves in the introduced range, but only 2x more highly defended in the native range. Additionally, defense levels are tracked by patterns of chewing damage, with damage restricted mostly to low-quality old leaves in the introduced range, but not the native range. Given that whole-plant investment in defense does not differ between ranges, introduced mullein may achieve increased fitness simply by optimizing its within-plant distribution of defense in the absence of certain specialist herbivores.

Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation.

An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and started growing when excised from mother explants. The transgenic nature of the most vigorously growing hairy root clones (VX1 and VX6) was confirmed by polymerase chain reaction. Under submerged cultivation both hairy root clones accumulated high biomass amounts (12.8 and 14.3 g L(-1), respectively) and significant amounts of bioactive phenylethanoid glycoside verbascoside (over 6-times more than in mother plant leaves). LC-APCI-MS analyses confirmed verbascoside accumulation in hairy root clones along with three other phenylethanoid glycosides (forsythoside B, leucosceptoside B and martynoside) and an iridoid glycoside aucubin. This is the first report on the induction of hairy roots of Verbascum plants.

Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading.

Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 (SUT1), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, and Verbascum phoeniceum, a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H(+) symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum, leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species.

Founding events in species invasions: genetic variation, adaptive evolution, and the role of multiple introductions.

Invasive species are predicted to suffer from reductions in genetic diversity during founding events, reducing adaptive potential. Integrating evidence from two literature reviews and two case studies, we address the following questions: How much genetic diversity is lost in invasions? Do multiple introductions ameliorate this loss? Is there evidence for loss of diversity in quantitative traits? Do invaders that have experienced strong bottlenecks show adaptive evolution? How do multiple introductions influence adaptation on a landscape scale? We reviewed studies of 80 species of animals, plants, and fungi that quantified nuclear molecular diversity within introduced and source populations. Overall, there were significant losses of both allelic richness and heterozygosity in introduced populations, and large gains in diversity were rare. Evidence for multiple introductions was associated with increased diversity, and allelic variation appeared to increase over long timescales (~100 years), suggesting a role for gene flow in augmenting diversity over the long-term. We then reviewed the literature on quantitative trait diversity and found that broad-sense variation rarely declines in introductions, but direct comparisons of additive variance were lacking. Our studies of Hypericum canariense invasions illustrate how populations with diminished diversity may still evolve rapidly. Given the prevalence of genetic bottlenecks in successful invading populations and the potential for adaptive evolution in quantitative traits, we suggest that the disadvantages associated with founding events may have been overstated. However, our work on the successful invader Verbascum thapsus illustrates how multiple introductions may take time to commingle, instead persisting as a 'mosaic of maladaptation' where traits are not distributed in a pattern consistent with adaptation. We conclude that management limiting gene flow among introduced populations may reduce adaptive potential but is unlikely to prevent expansion or the evolution of novel invasive behaviour.

Phloem loading in Verbascum phoeniceum L. depends on the synthesis of raffinose-family oligosaccharides.

Phloem loading is the initial step in photoassimilate export and the one that creates the driving force for mass flow. It has been proposed that loading occurs symplastically in species that translocate carbohydrate primarily as raffinose family oligosaccharides (RFOs). In these plants, dense fields of plasmodesmata connect bundle sheath cells to specialized companion cells (intermediary cells) in the minor veins. According to the polymer trap model, advanced as a mechanism of symplastic loading, sucrose from the mesophyll diffuses into intermediary cells and is converted there to RFOs. This process keeps the sucrose concentration low and, because of the larger size of the RFOs, prevents back diffusion. To test this model, the RFO pathway was down-regulated in Verbascum phoeniceum L. by suppressing the synthesis of galactinol synthase (GAS), which catalyzes the first committed step in RFO production. Two GAS genes (VpGAS1 and VpGAS2) were cloned and shown to be expressed in intermediary cells. Simultaneous RNAi suppression of both genes resulted in pronounced inhibition of RFO synthesis. Phloem transport was negatively affected, as evidenced by the accumulation of carbohydrate in the lamina and the reduced capacity of leaves to export sugars during a prolonged dark period. In plants with severe down-regulation, additional symptoms of reduced export were obvious, including impaired growth, leaf chlorosis, and necrosis and curling of leaf margins.