Molecular and Cytogenetic Analysis of rDNA Evolution in Crepis Sensu Lato.

Although Crepis was the first model plant group in which chromosomal changes were considered to play an important role in speciation, their chromosome structure and evolution have been barely investigated using molecular cytogenetic methods. The aim of the study was to provide a better understanding of the patterns and directions of Crepis chromosome evolution, using comparative analyses of rDNA loci number and localisation. The chromosome base number and chromosomal organisation of 5S and 35S rDNA loci were analysed in the phylogenetic background for 39 species of Crepis, which represent the evolutionary lineages of Crepis sensu stricto and Lagoseris, including Lapsana communis. The phylogenetic relationships among all the species were inferred from nrITS and newly obtained 5S rDNA NTS sequences. Despite high variations in rDNA loci chromosomal organisation, most species had a chromosome with both rDNA loci within the same (usually short) chromosomal arm. The comparative analyses revealed several independent rDNA loci number gains and loci repositioning that accompanied diversification and speciation in Crepis. Some of the changes in rDNA loci patterns were reconstructed for the same evolutionary lineages as descending dysploidy.

A Bio-Guided Screening for Antioxidant, Anti-Inflammatory and Hypolipidemic Potential Supported by Non-Targeted Metabolomic Analysis of Crepis spp.

This study was designed to evaluate the chemical fingerprints and the antioxidant, anti-inflammatory and hypolipidemic activity of selected Crepis species collected in Greece, namely, C. commutata, C. dioscoridis, C. foetida, C. heldreichiana, C. incana, C. rubra, and Phitosia crocifolia (formerly known as Crepis crocifolia). For the phytochemical analyses, sample measurements were carried out by using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS). Τhe extracts were evaluated both in vitro (radical scavenging activity: DPPH assay and total phenolic content: Folin-Ciocalteu) and in vivo (paw edema reduction and hypolipidemic activity: experimental mouse protocols). Among the tested extracts, C. incana presented the highest gallic acid equivalents (GAE) (0.0834 mg/mL) and the highest antioxidant activity (IC50 = 0.07 mg/mL) in vitro, as well as the highest anti-inflammatory activity with 32% edema reduction in vivo. Moreover, in the hypolipidemic protocol, the same extract increased plasma total antioxidant capacity (TAC) by 48.7%, and decreased cholesterol (41.3%) as well as triglycerides (37.2%). According to fractionation of the extract and the phytochemical results, this biological effect may be associated with the rich phenolic composition; caffeoyl tartaric acid derivatives (cichoric and caftaric acid) are regarded as the most prominent bioactive specialized metabolites. The present study contributes to the knowledge regarding the phytochemical and pharmacological profile of Crepis spp.

In vivo antiulcer activity, phytochemical exploration, and molecular modelling of the polyphenolic-rich fraction of Crepis sancta extract.

Bioactivity-guided investigation of the methanol extract of Crepis sancta aerial parts, collected off Al-Tafilah, South Jordan, was applied, and in this study, the extract was explored for its phytochemical components and in vivo antiulcer activity. In addition, a docking study involving the purified compounds with the newly crystalized gastric proton pump (PDB # 5YLU) was performed. In-depth phytochemical investigation using the state-of-the-art chromatographic and analytical techniques was implemented resulting in the identification of two eudesmane-type sesquiterpenoids, 3-oxo-γ-costic acid (1) and its methyl ester (2) together with seven different methoxylated flavonols (3-9) as the extract's major components. The in vivo antiulcer study at three different doses (50, 100, and 200 mg/kg) against ethanol-induced gastric ulcer in male albino rats, compared to omeprazole (20 mg/kg) as a standard proton pump inhibitor antiulcer drug, revealed that the tested extract, at the middle and the highest doses, featured comparable or even superior activities relative to omeprazole as deduced from histopathological examination, in particular with regard to reducing inflammatory cell infiltration and ceasing mucosal haemorrhage. The tested extract revealed also a dose-dependent reduction in the volume and titrable acidity of the gastric juice together with a dose-dependent increase in the protective gastric mucin content which may explain the noticeable gastroprotective effect. Molecular modelling study of the isolated compounds showed a binding mode similar to the co-crystallized substrate vonoprazan in 5YLU which strengthens the importance of the tested extract as a potential natural remedy for treating gastric ulcer.

[Anti-inflammatory mechanism of Crepis crocea based on NF-κB signaling pathway and ~1H-NMR metabonomics].

Based on ~1H-NMR metabonomics technique and Western blot assay, the anti-inflammatory mechanism of Crepis crocea was discussed. In this study, male SD rats were treated with water extract(2.5 g·kg~(-1)) and dexamethasone acetate(6.25×10~(-4) g·kg~(-1)) for one week, and the inflammation model was induced by lipopolysaccharide(LPS). Then the counts of inflammatory cells white blood ceel(WBC), eosinophil(EO), lymphocyte(LY), basophils(BA) and neutrophils(NE) in whole blood of rats were observed. The levels of serum inflammatory factors tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), IL-6 and the expression of nuclear factor-κB(NF-κB) signaling pathway p65 and p-IκBα proteins in lung tissues were detected, and the change rules of serum endogenous metabolites were analyzed by ~1H-NMR metabonomics technique. The levels of TNF-α, IL-1ß, IL-6 and NF-κB signaling pathway p65 and p-IκBα proteins were combined with ~1H-NMR metabonomics to study the anti-inflammatory mechanism of C. crocea. The results showed that the water extract of C. crocea significantly decreased the number of WBC, NE, EO, increased the number of BA and LY, decreased the levels of TNF-α, IL-1ß, IL-6 and the expression of p65 and p-IκBα protein in NF-κB signaling pathway, and effectively alleviated the inflammatory symptoms. In the correlation analysis of differential metabolites regulated of C. crocea, four significant metabolites were obtained, including glycine, creatine, methionine and succinic acid. The anti-inflammatory mechanism of C. crocea may be related to the decrease of TNF-α, IL-1ß, IL-6 levels and the protein expression of NF-κB signaling pathway, as well as the regulation of glycine, creatine, methionine and succinic acid metabolism.

Toxicity of Crepis lacera in grazing ruminants.

BACKGROUND: Crepis lacera is a plant from the Asteraceae family that is common in the Mediterranean region. Farmers believe that this plant may be deadly to small ruminants in areas of southern Italy. However, scientific evidence is lacking, and no proof exists that C. lacera is toxic to ruminants. Necropsies conducted on four sheep revealed lesions in their livers and kidneys. RESULTS: In the current study, we described sheep poisoning and isolated secondary metabolites from Crepis lacera to assess the metabolites' biological activity both in vitro and in vivo. Phytochemical study of the aerial portions of Crepis lacera led to the isolation of five sesquiterpene lactones and two phenolic compounds. Cellular viability was evaluated in cell cultures of the bovine kidney cell line Madin Darby Bovine Kidney (MDBK) after incubation with phytochemicals. Our results showed that three sesquiterpene lactones, 8-epidesacylcynaropicrin-3-O-ß-glucopyranoside (2), 8-epigrosheimin (3), and 8-ß-hydroxydehydrozaluzanin C (4), were cytotoxic after 48 h of incubation. In addition, in the in vivo study, animals that received 1 mg/kg body weight (bw) of Crepis lacera extract and were then sacrificed after 48 h showed significant lesions in their liver, lungs and kidneys. These lesions were also found in rats that received 2 mg/kg bw of the same extract and sacrificed after 24 and 48 h. CONCLUSIONS: These results validate the hypothesis that C. lacera is potentially dangerous when ingested in large quantities by grazing small domestic ruminants. Further studies are necessary to clarify the molecular mechanisms of Crepis spp. toxicity in animals.

Responses of Crepis japonica induced by supplemental blue light and UV-A radiation.

Crepis japonica (L.) D.C. (Asteraceae), a weed with antioxidant, antiallergenic, antiviral and antitumor properties displays both medicinal properties and nutritional value. This study aims to assess the effects of a supplementation of blue light and UV-A radiation on the growth, leaf anatomical structure and phenolic profile of the aerial parts of Crepis japonica. Plants were grown under two light treatments: W (control - white light), W + B (white light supplemented with blue light) and W + UV-A (white light supplemented with UV-A radiation). We recorded the length, width, and weight of fresh and dry leaves, the thickness of the epidermis and mesophyll, and stomata density. The phenolic profiles of the aqueous extracts of the aerial parts were analyzed by HPLC-DAD. There was an increase in the leaf size, stomatal density, and phenolic production, and a thickening of the mesophyll and epidermis. UV-A radiation increased the phenolic production more than blue light. Blue light and UV-A radiation both improved the production of caffeic acid by about 6 and 3 times, respectively, in comparison to control. This compound was first reported as a constituent of the extract from the aerial parts together with caftaric acid. UV-A also promoted the production of chlorogenic acid (about 1.5 times in comparison to the control). We observed that the morphological and chemical parameters of C. japonica are modified in response to blue light and UV-A radiation, which can be used as tools in the cultivation of this species in order to improve its medicinal properties and nutritional value.

Is it worth hyperaccumulating Ni on non-serpentine soils? Decomposition dynamics of mixed-species litters containing hyperaccumulated Ni across serpentine and non-serpentine environments.

BACKGROUND AND AIMS: Nickel (Ni)-hyperaccumulating species produce high-Ni litters and may potentially influence important ecosystem processes such as decomposition. Although litters resembling the natural community conditions are essential in order to predict decomposition dynamics, decomposition of mixed-species litters containing hyperaccumulated Ni has never been studied. This study aims to test the effect of different litter mixtures containing hyperaccumulated Ni on decomposition and Ni release across serpentine and non-serpentine soils. METHODS: Three different litter mixtures were prepared based on the relative abundance of the dominant species in three serpentine soils in the island of Lesbos, Greece where the Ni-hyperaccumulator Alyssum lesbiacum is present. Each litter mixture decomposed on its original serpentine habitat and on an adjacent non-serpentine habitat, in order to investigate whether the decomposition rates differ across the contrasted soils. In order to make comparisons across litter mixtures and to investigate whether additive or non-additive patterns of mass loss occur, a control non-serpentine site was used. Mass loss and Ni release were measured after 90, 180 and 270 d of field exposure. KEY RESULTS: The decomposition rates and Ni release had higher values on serpentine soils after all periods of field exposure. The recorded rapid release of hyperaccumulated Ni is positively related to the initial litter Ni concentration. No differences were found in the decomposition of the three different litter mixtures at the control non-serpentine site, while their patterns of mass loss were additive. CONCLUSIONS: Our results: (1) demonstrate the rapid decomposition of litters containing hyperaccumulated Ni on serpentine soils, indicating the presence of metal-tolerant decomposers; and (2) imply the selective decomposition of low-Ni parts of litters by the decomposers on non-serpentine soils. This study provides support for the elemental allelopathy hypothesis of hyperaccumulation, presenting the potential selective advantages acquired by metal-hyperaccumulating plants through litter decomposition on serpentine soils.

A reexamination of the North American Crepis agamic complex and comparison with the findings of Babcock and Stebbins' classic biosystematic monograph.

PREMISE OF THE STUDY: Babcock and Stebbins coined the term agamic complex in their 1938 monograph of the North American Crepis agamic complex. Despite the historical role that this complex holds in the evolutionary literature, it has not been reexamined in over 75 years. We present a thorough reevaluation of the complex to test hypotheses proposed by Babcock and Stebbins about its origins and spread, the relationships of diploids, and the nature and origins of polyploids. METHODS: We used flow cytometry to infer ploidy of roughly 600 samples spanning the morphological and taxonomic diversity of the complex and a phylogenetic analysis of plastid DNA variation to infer maternal relationships among diploids and to infer maternal origins of polyploids. KEY RESULTS: We identified populations of all seven recognized diploids plus one new lineage. Phylogenetic analysis of plastid DNA variation in diploids revealed a well-resolved, but moderately supported phylogeny, with evidence for monophyly of the North America Crepis agamic complex and no evidence of widespread homoploid hybridization. Polyploids showed evidence of multiple origins and a pattern of frequent local co-occurrence consistent with repeated colonization of suitable sites. CONCLUSIONS: Our findings agree broadly with the distribution and variation of ploidy within and among species described by Babcock and Stebbins. One key difference is finding support for monophyly of North American species, and refuting their hypothesis of polyphyly. Our results provide an explicit phylogenetic framework for further study of this classic agamic complex.

[Chemical constituents from Crepis crocea].

Thirteen compounds were isolated from the ethyl acetate fraction of Crepis crocea by column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. The structures were elucidated on the basis of spectral analysis as tectorone I (1), 8ß- (2-methyl- 2-hydroxy-3-oxobutanoyloxy) -glucozaluzanin C (2), tectoroside (3), luteolin-7-O-glucoside (4), cosmosiin (5), esculetin (6), 3,4-dihydroxybenzaldehyde (7), trans-4-hydroxycinnamic acid (8), Caffeic acid (9), methyl p-hydroxyphenyllactate (10), ethylp- hydroxyphenyllactate (11), cis-3,4-dihydroxy-ß-ionion (12). All the compounds, except for compounds 4 and 9, were isolated from this plant for the first time, and tectorone I (1) is a new natural product.

Inferring contemporary dispersal processes in plant metapopulations: comparison of direct and indirect estimates of dispersal for the annual species Crepis sancta.

Analyzing population dynamics in changing habitats is a prerequisite for population dynamics forecasting. The recent development of metapopulation modeling allows the estimation of dispersal kernels based on the colonization pattern but the accuracy of these estimates compared with direct estimates of the seed dispersal kernel has rarely been assessed. In this study, we used recent genetic methods based on parentage analysis (spatially explicit mating models) to estimate seed and pollen dispersal kernels as well as seed and pollen immigration in fragmented urban populations of the plant species Crepis sancta with contrasting patch dynamics. Using two independent networks, we documented substantial seed immigration and a highly restricted dispersal kernel. Moreover, immigration heterogeneity among networks was consistent with previously reported metapopulation dynamics, showing that colonization was mainly due to external colonization in the first network (propagule rain) and local colonization in the second network. We concluded that the differences in urban patch dynamics are mainly due to seed immigration heterogeneity, highlighting the importance of external population source in the spatio-temporal dynamics of plants in a fragmented landscape. The results show that indirect and direct methods were qualitatively consistent, providing a proper interpretation of indirect estimates. This study provides attempts to link genetic and demographic methods and show that patch occupancy models may provide simple methods for analyzing population dynamics in heterogeneous landscapes in the context of global change.

Effects of mating system on adaptive potential for leaf morphology in Crepis tectorum (Asteraceae).

BACKGROUND AND AIMS: A shift from outcrossing to selfing is thought to reduce the long-term survival of populations by decreasing the genetic variation necessary for adaptation to novel ecological conditions. However, theory also predicts an increase in adaptive potential as more of the existing variation becomes expressed as homozygous genotypes. So far, relatively few studies have examined how a transition to selfing simultaneously affects means, variances and covariances for characters that might be under stabilizing selection for a spatially varying optimum, e.g. characters describing leaf morphology. METHODS: Experimental crosses within an initially self-sterile population of Crepis tectorum were performed to produce an outbred and inbred progeny population to assess how a shift to selfing affects the adaptive potential for measures of leaf morphology, with special emphasis on the degree of leaf dissection, a major target of diversifying selection within the study species. KEY RESULTS: Three consecutive generations of selfing had a minor impact on survival, the total number of heads produced and the mean leaf phenotype, but caused a proportional increase in the genetic (co)variance matrix for foliar characters. For the degree of leaf dissection, the lowest 50th percentile of the inbred progeny population showed a disproportionate increase in the genetic variance, consistent with the recessive nature of the weakly lobed phenotype observed in interpopulation crosses. Comparison of inbreeding response with large-scale patterns of variation indicates a potential for selection in a (recently) inbred population to drive a large evolutionary reduction in degree of leaf dissection by increasing the frequency of particular sibling lines. CONCLUSIONS: The results point to a positive role for inbreeding in phenotypic evolution, at least during or immediately after a rapid shift in mating system.

Clastogenic and anticlastogenic activity of glucocorticoid hormones hydrocortisone and its synthetic analogs prednisolone and dexamethasone.

Clastogenic and anticlastogenic activity of glucocorticoid hormones hydrocortisone, prednisolone, and dexamethasone was studied by counting chromosome aberrations in Crepis capillaris test system. Hydrocortisone in a concentration of 12.5 mg/ml produced a clastogenic effect and increased the number of chromosome aberrations in comparison with spontaneous level. Hydrocortisone (6.25 and 3.13 mg/ml), prednisolone (15, 7.5, and 3.75 mg/ml), and dexamethasone (1, 0.5, 0.25, and 0.125 mg/ml) exhibited an anticlastogenic effect and reduced ethyl methanesulfonate-induced mutagenesis.

Competition/colonization syndrome mediated by early germination in non-dispersing achenes in the heteromorphic species Crepis sancta.

BACKGROUND AND AIMS: The competition-colonization trade-off theory postulates that the competitive and colonizing abilities of organisms are negatively related; this trade-off has been proposed as a major force in the maintenance of diversity. In plants, the competition-colonization trade-off is often considered to result from variation in resource partitioning, thus generating heavy competitive (non-dispersing) seeds and light (dispersing) non-competitive seeds. Here, the possibility is explored that early germination provides a competitive advantage, thus mediating competitive interactions. METHODS: Using eight populations of the heterocarpic species Crepis sancta (Asteraceae), the possibility was tested that dispersing and non-dispersing achenes differ in germination timing, and the impact of early germination on individual fitness components was analysed in the context of intraspecific competition. To evaluate whether seed reserve varies among achene types, endosperm size was also measured by analysing photographs of cross-sections taken under a binocular microscope. KEY RESULTS AND CONCLUSIONS: The results show that non-dispersing achenes germinated 4 d earlier (on average) than dispersing achenes. It is also shown that early germination provides a positive advantage for the survival and final biomass of individuals, a pattern that was consistent over the eight populations and independent of achene type. Dispersing and non-dispersing achenes did not differ in terms of seed reserve (endosperm size). It is proposed that germination phenology may mediate the competition-colonization trade-off in Crepis sancta and the evolutionary significance of this phenomenon is discussed.

Patterns of genetic variability and habitat occupancy in Crepis triasii (Asteraceae) at different spatial scales: insights on evolutionary processes leading to diversification in continental islands.

BACKGROUND AND AIMS: Archipelagos are unique systems for studying evolutionary processes promoting diversification and speciation. The islands of the Mediterranean basin are major areas of plant richness, including a high proportion of narrow endemics. Many endemic plants are currently found in rocky habitats, showing varying patterns of habitat occupancy at different spatial scales throughout their range. The aim of the present study was to understand the impact of varying patterns of population distribution on genetic diversity and structure to shed light on demographic and evolutionary processes leading to population diversification in Crepis triasii, an endemic plant from the eastern Balearic Islands. METHODS: Using allozyme and chloroplast markers, we related patterns of genetic structure and diversity to those of habitat occupancy at a regional (between islands and among populations within islands) and landscape (population size and connectivity) scale. KEY RESULTS: Genetic diversity was highly structured both at the regional and at the landscape level, and was positively correlated with population connectivity in the landscape. Populations located in small isolated mountains and coastal areas, with restricted patterns of regional occupancy, were genetically less diverse and much more differentiated. In addition, more isolated populations had stronger fine-scale genetic structure than well-connected ones. Changes in habitat availability and quality arising from marine transgressions during the Quaternary, as well as progressive fragmentation associated with the aridification of the climate since the last glaciation, are the most plausible factors leading to the observed patterns of genetic diversity and structure. CONCLUSIONS: Our results emphasize the importance of gene flow in preventing genetic erosion and maintaining the evolutionary potential of populations. They also agree with recent studies highlighting the importance of restricted gene flow and genetic drift as drivers of plant evolution in Mediterranean continental islands.

Does inbreeding promote evolutionary reduction of flower size? Experimental evidence from Crepis tectorum (Asteraceae).

PREMISE OF THE STUDY: Small, autogamous flowers have evolved repeatedly in the plant kingdom. While much attention has focused on the mechanisms that promote the shift to autogamy, there is still a paucity of information on the factors that underlie the reduction of flower size so prevalent in selfing lineages. In this study of Crepis tectorum, I examine the role of inbreeding, acting alone or together with selection, in promoting evolutionary reduction of flower size. METHODS: Experimental crosses were performed to produce progeny populations that differed in inbreeding and (or) selection history. Progenies were grown in two different environments and scored for flower size and other characters. KEY RESULTS: Inbreeding depressed flower and fruit size, but also caused changes in flowering time and the number of heads produced. Despite some inconsistencies in the results for the last progeny generation, the decline in flower size was persistent over generations, consistent across environments, and similar in magnitude to the effects of selection for small flower size and the floral reduction inferred to have taken place during the shift toward autogamy within the study species. The floral size reduction was largely independent of changes in overall vigor, and there was considerable adaptive potential in flower size (measured by sib analyses and parent-offspring comparisons) after inbreeding. CONCLUSIONS: The results of this study indicate that inbreeding can promote evolutionary reduction of flower size and highlight the close, persistent association between flower and fruit size in the study species.

Determinants of extinction in fragmented plant populations: Crepis sancta (Asteraceae) in urban environments.

Local populations are subject to recurrent extinctions, and small populations are particularly prone to extinction. Both demographic (stochasticity and the Allee effect) and genetic factors (drift load and inbreeding depression) potentially affect extinction. In fragmented populations, regular dispersal may boost population sizes (demographic rescue effect) or/and reduce the local inbreeding level and genetic drift (genetic rescue effect), which can affect extinction risks. We studied extinction processes in highly fragmented populations of the common species Crepis sancta (Asteraceae) in urban habitats exhibiting a rapid turnover of patches. A four-year demographic monitoring survey and microsatellite genotyping of individuals allowed us to study the determinants of extinction. We documented a low genetic structure and an absence of inbreeding (estimated by multilocus heterozygosity), which suggest that genetic factors were not a major cause of patch extinction. On the contrary, local population size was the main factor in extinction, whereas connectivity was shown to decrease patch extinction, which we interpreted as a demographic rescue effect that was likely due to better pollination services for reproduction. This coupling of demographic and genetic tools highlighted the importance of dispersal in local patch extinctions of small fragmented populations connected by gene flow.

Linné's floral clock is slow without pollinators--flower closure and plant-pollinator interaction webs.

Temporal patterns of flower opening and closure within a day are known as Linné's floral clock. Time of flower closure has been explained mainly by light in the traditional botanical literature. We show with a set of experiments that Asteraceae flower heads can close within three hours after pollination, whereas un-pollinated flower heads stay open until the late afternoon. This suggests that closing time strongly depends on pollinators. Using plant-pollinator interaction webs we further demonstrate that the daily pattern of flower opening and the rapid response to pollination can impose strong temporal dynamics on interspecific interactions within a single day. We observed pollinator species turnover and changes in facilitation vs. competition among plants. Our results show for the first time that pollination induces rapid flower closure on the community level. This causes imprecision in Linné's floral clock with far-reaching consequences for plant-pollinator interactions.

Rapid evolution of seed dispersal in an urban environment in the weed Crepis sancta.

Dispersal is a ubiquitous trait in living organisms. Evolutionary theory postulates that the loss or death of propagules during dispersal episodes (cost of dispersal) should select against dispersal. The cost of dispersal is expected to be a strong selective force in fragmented habitats. We analyzed patchy populations of the weed Crepis sancta occupying small patches on sidewalks, around trees planted within the city of Montpellier (South of France), to investigate the recent evolutionary consequences of the cost of dispersal. C. sancta produces both dispersing and nondispersing seeds. First, we showed that, in urban patches, dispersing seeds have a 55% lower chance of settling in their patch compared with nondispersing seeds and, thus, fall on a concrete matrix unsuitable for germination. Second, we showed that the proportion of nondispersing seeds in urban patches measured in a common environment is significantly higher than in surrounding, unfragmented populations. Third, by using a quantitative genetic model, we estimated that the pattern is consistent with short-term evolution that occurs over approximately 5-12 generations of selection, which is generated by a high cost of dispersal in urban populations. This study shows that a high cost of dispersal after recent fragmentation causes rapid evolution toward lower dispersal.

[Determination of the mutagenic potential of inorganic compounds of some heavy metals].

The authors determined the mutagenic potential of inorganic compounds, such as tungsten, molybdenum, lead, and copper, as well as waste of a tungsten-molybdenum industrial complex engaged in the exploitation of tungsten and molybdenum deposits, and environmental components, by using a plant test system and species of wild flora. They were shown to be suitable for the determination of genotoxicity of the environment polluted with heavy metals.