Effects of two substrates at different phosphorus levels on morphology and physiology of Dianthus barbatus Linn.

Dianthus barbatus linn. is widely used in gardens, mainly as flower beds and flower borders. The effects of different gradients of P on the growth and root morphology of Dianthus barbatus were studied to explore its morphological and physiological responses and adaptive strategies. Hence, this study provides a theoretical basis and practical guidance for D. barbatus production. Two soil substrates, namely loess and vegetable soil, and five phosphorus concentration gradients were set; no phosphorus application was used as the control. The morphology and physiology of D. barbatus were also investigated. Low-to-medium- and low-phosphorus treatments promoted the growth of D. barbatus in the above and underground parts of the plants grown on both substrates. Chlorophyll content, flower quantity, and acid phosphatase activity in the rhizosphere soil were significantly increased in the H1 and H2 treatments of loess and in the C4 treatment of vegetable soil. Thus, D. barbatus seems to reduce the damage caused by phosphorus stress by increasing chlorophyll content and root acid phosphatase activity. The latter was significantly higher in vegetable soil than in loess. Vegetable soil was more conducive to D. barbatus growth than loess.

DcWRKY75 promotes ethylene induced petal senescence in carnation (Dianthus caryophyllus L.).

Carnation (Dianthus caryophyllus L.) is one of the most important and typical ethylene sensitive cut flowers worldwide, although how ethylene influences the petal senescence process in carnation remains largely unknown. Here, we screened out one of the key transcription factors, DcWRKY75, using a constructed ethylene induced petal senescence transcriptome in carnation and found that it shows quick induction by ethylene treatment. Silencing of DcWRKY75 delays ethylene induced petal senescence in carnation. Molecular evidence confirms that DcWRKY75 can bind to the promoter regions of two main ethylene biosynthetic genes (DcACS1 and DcACO1) and a couple of senescence associated genes (DcSAG12 and DcSAG29) to activate their expression. Furthermore, we show that DcWRKY75 is a direct target gene of DcEIL3-1, which is a homolog of the ethylene signaling core transcription factor EIN3 in Arabidopsis. DcEIL3-1 can physically interact with DcWRKY75 and silencing of DcEIL3-1 also delays ethylene induced petal senescence in carnation and inhibits the ethylene induced expression of DcWRKY75 and its target genes. The present study demonstrates that the transcriptional regulation network is vitally important for ethylene induced petal senescence process in carnation and potentially in other ethylene sensitive cut flowers.

Soil chemical legacies trigger species-specific and context-dependent root responses in later arriving plants.

Soil legacies play an important role for the creation of priority effects. However, we still poorly understand to what extent the metabolome found in the soil solution of a plant community is conditioned by its species composition and whether soil chemical legacies affect subsequent species during assembly. To test these hypotheses, we collected soil solutions from forb or grass communities and evaluated how the metabolome of these soil solutions affected the growth, biomass allocation and functional traits of a forb (Dianthus deltoides) and a grass species (Festuca rubra). Results showed that the metabolomes found in the soil solutions of forb and grass communities differed in composition and chemical diversity. While soil chemical legacies did not have any effect on F. rubra, root foraging by D. deltoides decreased when plants received the soil solution from a grass or a forb community. Structural equation modelling showed that reduced soil exploration by D. deltoides arose via either a root growth-dependent pathway (forb metabolome) or a root trait-dependent pathway (grass metabolome). Reduced root foraging was not connected to a decrease in total N uptake. Our findings reveal that soil chemical legacies can create belowground priority effects by affecting root foraging in later arriving plants.

Flower color mutation caused by spontaneous cell layer displacement in carnation (Dianthus caryophyllus).

A change of layer arrangement of shoot apical meristem (SAM) organized by three cell layers (L1, L2 and L3) is thought to be one of the provocations of bud sport, which often induces changes in phenotypic colors in periclinal chimeras. This paper describes a cell layer rearrangement which is the cause of spontaneous flower color mutation by using two carnation (Dianthus caryophyllus L.) cultivars that are presumably periclinal chimeras, 'Feminine Minami' (deep pink flower) and its recessive sport 'Tommy Minami' (pinkish red flower). The genotype of the acyl-glucose-dependent anthocyanin 5-glucosyltransferase (AA5GT) which is responsible for the color change of red to pink, in each cell layer was deduced by genomic analysis using tissues originated from specific cell layer and investigation of partial petal color mutations. In the results, the genotype of the L1 of 'Feminine Minami' was heterozygous for functional AA5GT and non-functional AA5GT carrying retrotransposon Ty1dic1 (AA5GT-Ty1dic1), and its inner cell layer hid red flower genotype, whereas AA5GT-Ty1dic1 of the L1 of 'Tommy Minami' became homogenic in absence of the insertion of a new Ty1dic1. Our outcomes concluded that the L1 of 'Tommy Minami' harboring the recessive AA5GT alleles are attributed to the inner cell layer of 'Feminine Minami' possessing red flower genotype.

Climate change influences on the potential distribution of Dianthus polylepis Bien. ex Boiss. (Caryophyllaceae), an endemic species in the Irano-Turanian region.

Endemic and restricted-range species are considered to be particularly vulnerable to the effects of environmental change, which makes assessing likely climate change effects on geographic distributions of such species important to the development of integrated conservation strategies. Here, we determined distributional patterns for an endemic species of Dianthus (Dianthus polylepis) in the Irano-Turanian region using a maximum-entropy algorithm. In total, 70 occurrence points and 19 climatic variables were used to estimate the potential distributional area under current conditions and two future representative concentration pathway (RCP2.6 and RCP8.5) scenarios under seven general circulation models for 2050. Mean diurnal range, iso-thermality, minimum temperature of coldest quarter, and annual precipitation were major factors that appeared to structure the distribution of the species. Most current potential suitable areas were located in montane regions. Model transfers to future-climate scenarios displayed upward shifts in elevation and northward shifts geographically for the species. Our results can be used to define high-priority areas in the Irano-Turanian region for conservation management plans for this species and can offer a template for analyses of other endangered and threatened species in the region.

Blue light postpones senescence of carnation flowers through regulation of ethylene and abscisic acid pathway-related genes.

Endogenous signals in response to exogenous factors determine the senescence of flowers. Interactions among phytohormones especially abscisic acid (ABA) and ethylene are the major determinant of the senescence. In the present study, complex expression patterns of the genes related to ABA and ethylene as endogenous signals were investigated on cut carnations (Dianthus caryophyllus L.) that were exposed to different light spectra. Expression of ethylene biosynthetic (DcACS and DcACO), and signaling (DcETR and DcEin2) genes and also genes involved in ABA biosynthesis (DcZEP1 and DcNCED1), transport (DcABCG25 and DcABCG40) and catabolism (DcCYP707A1) were evaluated in petals of carnations exposed to three light spectra [white, blue and red]. Lowest relative membrane permeability (RMP) was detected in flowers that exposed to Blue light (BLFs), as a consequence, the longest vase life was found in BLFs. The Red and White lights markedly accelerated flower senescence and increased expression of DcACS and DcACO on day 6 and 10 of vase life assessment respectively; while Blue light inhibited the expression of ethylene biosynthetic genes. Expression of the genes involved in the production and transport of ABA and in signal transduction of ethylene was elevated during vase life of flowers irrespective of exposure to different light spectra. In conclusion, Blue light can be an effective environmental factor to extend the vase life of carnation flowers by delaying the petal senescence through down-regulation of ethylene biosynthetic genes and up-regulation of ABA biosynthetic genes.

The role of infectious disease in the evolution of females: Evidence from anther-smut disease on a gynodioecious alpine carnation.

In flowering plants, the evolution of females is widely hypothesized to be the first step in the evolutionary pathway to separate male and female sexes, or dioecy. Natural enemies have the potential to drive this evolution if they preferentially attack hermaphrodites over females. We studied sex-based differences in exposure to anther-smut (Microbotryum), a sterilizing pollinator-transmitted disease, in Dianthus pavonius, a gynodioecious perennial herb. We found that within a heavily diseased population, females consistently had lower levels of Microbotryum spore deposition relative to hermaphrodites and that this difference was driven by rapid floral closing in females following successful pollination. We further show that this protective closing behavior is frequency dependent; females close faster when they are rare. These results indicate that anther-smut disease is an important source of selection for females, especially since we found in a common garden experiment no evidence that females have any inherent fecundity advantages over hermaphrodites. Finally, we show that among populations, those where anther-smut is present have a significantly higher frequency of females than those where the disease is absent. Taken together our results indicate that anther-smut disease is likely an important biotic factor driving the evolution and maintenance of females in this gynodioecious species.

Divergent strategies in pre- and postzygotic reproductive isolation between two closely related Dianthus species.

Quantifying the relative contribution of multiple isolation barriers to gene flow between recently diverged species is essential for understanding speciation processes. In parapatric populations, local adaptation is thought to be a major contributor to the evolution of reproductive isolation. However, extrinsic postzygotic barriers assessed in reciprocal transplant experiments are often neglected in empirical assessments of multiple isolation barriers. We analyzed multiple isolation barriers between two closely related species of the plant genus Dianthus, a genus characterized by the most rapid species diversification in plants reported so far. Although D. carthusianorum L. and D. sylvestris Wulf. can easily be hybridized in crossing experiments, natural hybrids are rare. We found that in parapatry, pollinator-mediated prezygotic reproductive isolation barriers are important for both D. carthusianorum (0.761) and D. sylvestris (0.468). In contrast to D. carthusianorum, high hybrid viability in D. sylvestris (-0.491) was counteracted by strong extrinsic postzygotic isolation (0.900). Our study highlights the importance of including reciprocal transplant experiments for documenting extrinsic postzygotic isolation and demonstrates clearly divergent strategies and hence asymmetric pre- and postzygotic reproductive isolation between closely related species. It also suggests that pollinator-mediated and ecological isolation could have interacted in synergistic ways, further stimulating rapid speciation in Dianthus.

Characterization of the role of sodium nitroprusside (SNP) involved in long vase life of different carnation cultivars.

BACKGROUND: Sodium nitroprusside (SNP) has been previously shown to extend the vase life of various cut flowers; however, its positive effect on extending vase life of carnations has not been well documented. Moreover, the role of SNP in the mechanisms underlying determination of vase life of cut carnations has also not been well addressed. RESULTS: SNP increased vase life of Tico Viola carnations along with their relative fresh weight (RFW). Among the treatments, the flowers treated with 10 mg L-1 SNP had the longest vase life and maximum relative fresh weight (RFW). This was achieved through significant suppression of ethylene production via downregulation of ethylene biosynthesis and petal senescence-related genes, and through an increase in the scavenging mechanism of reactive oxygen species (ROS) by antioxidant activity during flower vase life. In addition, the positive efficacy of SNP could also be confirmed using 1-aminocyclopropane-1-carboxylic acid (ACC) and different cultivars, resulting in similar trends for both experiments. CONCLUSION: Taken together, these results suggest that SNP plays a crucial role in multiple modes of action that are associated with the longevity of cut carnation flowers.

Transcriptome Sequencing of Dianthus spiculifolius and Analysis of the Genes Involved in Responses to Combined Cold and Drought Stress.

Dianthus spiculifolius, a perennial herbaceous flower and a member of the Caryophyllaceae family, has strong resistance to cold and drought stresses. To explore the transcriptional responses of D. spiculifolius to individual and combined stresses, we performed transcriptome sequencing of seedlings under normal conditions or subjected to cold treatment (CT), simulated drought treatment (DT), or their combination (CTDT). After de novo assembly of the obtained reads, 112,015 unigenes were generated. Analysis of differentially expressed genes (DEGs) showed that 2026, 940, and 2346 genes were up-regulated and 1468, 707, and 1759 were down-regulated in CT, DT, and CTDT samples, respectively. Among all the DEGs, 182 up-regulated and 116 down-regulated genes were identified in all the treatment groups. Analysis of metabolic pathways and regulatory networks associated with the DEGs revealed overlaps and cross-talk between cold and drought stress response pathways. The expression profiles of the selected DEGs in CT, DT, and CTDT samples were characterized and confirmed by quantitative RT-PCR. These DEGs and metabolic pathways may play important roles in the response of D. spiculifolius to the combined stress. Functional characterization of these genes and pathways will provide new targets for enhancement of plant stress tolerance through genetic manipulation.

Reversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO3 and its associated mechanism during in vitro culture.

Hyperhydricity occurs frequently in plant tissue culture and can severely affect commercial micropropagation and genetic improvement of the cultured plantlets. Hyperhydric shoots are charaterzized by high water content, but how this occurs is still a subject of investigation. Silver ion (Ag+) can reduce the extent of hyperhydricity in plants, but its effect on the reversion of hyperhydric plantlets and the underlying mechanism of reversion has not been clarified. In this study, about 67% of the hyperhydric Dianthus chinensis L. plantlets were found to revert to normal condition when the plantlets were cultured in medium supplemented with 29.4µmolL-1AgNO3. Water content and hydrogen peroxide (H2O2) content in the guard cells of these plantlets were reduced, while stomatal aperture and water loss rate were increased. AgNO3 also reduced the content of endogenous ethylene and expression of ethylene synthesis and ethylene signal transduction-associated genes. Reduced accumulation of ethylene consequently led to an increase in stomatal aperture mediated by decreased H2O2 content in the guard cells. These results adequately verified the role of AgNO3 in the reversion of hyperhydricity in D. chinensis L. and also provided clues for exploring the cause of excessive water accumulation in hyperhydric plants.

Deciphering the ecophysiological traits involved during water stress acclimation and recovery of the threatened wild carnation, Dianthus inoxianus.

Dianthus inoxianus is an endangered species endemic from a small littoral area in the SW Spain, with an unusual flowering season under the adverse conditions of dry Mediterranean summer. A greenhouse experiment was designed to assess the physiological traits involved in drought acclimation and recovery of 3-month-old plants. The evolution of plant water status, leaf gas exchange, chlorophyll fluorescence, photosynthetic pigments concentrations and a quantitative analysis of photosynthesis limitations were followed during water stress and re-watering. Our results indicated that the plant water status, Ψw and RWC, only decreased at the end of the drought period (18th day), together with the net photosynthetic rate, AN. Photosynthetic impair was mainly caused by diffusional limitations (SL and MCL) of CO2, as indicated the joint and marked decrease of gs, gm and Ci during drought period, while Vc,max did not vary. After rewatering, leaf water status recovered faster than photosynthetic one, reaching control values on day 1 after recovery, while AN, gm and Ci took 7 days. Additionally, gs showed the slowest recovery taking 15 days, but gs decrease was enough to keep Ψw and RWC at constant values throughout the experiment. Results suggest a high tolerance and recovery of D. inoxianus from severe drought periods. This drought tolerance was also reflected in the stability of its photochemical apparatus and pigments concentrations, as indicated the constant values of Fv/Fm, ФPSII and pigments concentrations through experimental period. However, prolonged drought events due to global climate change could negatively affect the physiological mechanisms of this species.

Quantitative Analysis of Adventitious Root Growth Phenotypes in Carnation Stem Cuttings.

Carnation is one of the most important species on the worldwide market of cut flowers. Commercial carnation cultivars are vegetatively propagated from terminal stem cuttings that undergo a rooting and acclimation process. For some of the new cultivars that are being developed by ornamental breeders, poor adventitious root (AR) formation limits its commercial scaling-up, due to a significant increase in the production costs. We have initiated a genetical-genomics approach to determine the molecular basis of the differences found between carnation cultivars during adventitious rooting. The detailed characterization of AR formation in several carnation cultivars differing in their rooting losses has been performed (i) during commercial production at a breeders' rooting station and (ii) on a defined media in a controlled environment. Our study reveals the phenotypic signatures that distinguishes the bad-rooting cultivars and provides the appropriate set-up for the molecular identification of the genes involved in AR development in this species.

Identification of genes associated with chlorophyll accumulation in flower petals.

Plants have an ability to prevent chlorophyll accumulation, which would mask the bright flower color, in their petals. In contrast, leaves contain substantial amounts of chlorophyll, as it is essential for photosynthesis. The mechanisms of organ-specific chlorophyll accumulation are unknown. To identify factors that determine the chlorophyll content in petals, we compared the expression of genes related to chlorophyll metabolism in different stages of non-green (red and white) petals (very low chlorophyll content), pale-green petals (low chlorophyll content), and leaves (high chlorophyll content) of carnation (Dianthus caryophyllus L.). The expression of many genes encoding chlorophyll biosynthesis enzymes, in particular Mg-chelatase, was lower in non-green petals than in leaves. Non-green petals also showed higher expression of genes involved in chlorophyll degradation, including STAY-GREEN gene and pheophytinase. These data suggest that the absence of chlorophylls in carnation petals may be caused by the low rate of chlorophyll biosynthesis and high rate of degradation. Similar results were obtained by the analysis of Arabidopsis microarray data. In carnation, most genes related to chlorophyll biosynthesis were expressed at similar levels in pale-green petals and leaves, whereas the expression of chlorophyll catabolic genes was higher in pale-green petals than in leaves. Therefore, we hypothesize that the difference in chlorophyll content between non-green and pale-green petals is due to different levels of chlorophyll biosynthesis. Our study provides a basis for future molecular and genetic studies on organ-specific chlorophyll accumulation.

Ethylene production associated with petal senescence in carnation flowers is induced irrespective of the gynoecium.

To clarify whether climacteric-like increases in ethylene production of senescing petals are also induced in the absence of the gynoecium in cut carnation (Dianthus caryophyllus cv. Barbara) flowers, we compared ethylene production and expression of ethylene-biosynthesis genes in detached petals and in petals, which remained on flowers (attached petals). No significant difference in longevity was observed between the attached and detached petals when held in distilled water, and both showed the inward rolling typical of senescing flowers. Treatment with silver thiosulfate complex (STS), an ethylene inhibitor, similarly delayed senescence of attached and detached petals. Climacteric-like increases in ethylene production of petals and gynoecium started on the same day, with similar bursts in attached and detached petals. Transcript levels of DcACS1 and DcACO1 were very low at harvest and increased similarly during senescence in both petal groups. Removal of the gynoecium did not significantly delay wilting of attached petals. In flowers with the gynoecium removed, the petals produced most of the ethylene while production by the other floral organs was very low, suggesting that wound-induced ethylene is not the reason for the ineffectiveness of gynoecium-removal in inhibiting flower senescence. These results indicate that ethylene biosynthesis is induced in carnation petals irrespective of the gynoecium.

The role of silver nano-particles and silver thiosulfate on the longevity of cut carnation (Dianthus caryophyllus) flowers.

The purpose of this study was to evaluate the efficacy of silver nano-particles (SNP) and silver thiosulfate (STS) in extending the vase life of cut carnation (Dianthus caryophyllus L. cv. 'Tempo') flowers. Pulse treatments of SNP @ 0, 5, 10 and 15 mg l(-1) and STS @ 0, 0.1, 0.2 and 0.3 mM were administered to carnation flowers for 24 hr. The longest vase life (16.1 days) was observed in flowers treated with 15 mg l(-1) of SNP + 0.2 mM STS. The least chlorophyll was destroyed in flowers treated with 15 mg I(-1) of SNP + 0.3 mM STS. Our findings showed that the 15 mg l(-1) SNP treatment inhibited bacterial growth in the preservative solution. The control flowers bloomed faster than the treated flowers. The maximum peroxidase activity and the minimum lipid peroxidation were obtained in cut flowers that were treated with 15 mg l(-1) of SNP and 0.3 mM STS. Overall, results of the study revealed that SNP and STS treatment extended the longevity of cut carnation 'Tempo' flowers by reducing oxidative stress, improving anti-oxidant system, reducing bacterial populations and delaying flowering.

Accumulation and tolerance of lead in two contrasting ecotypes of Dianthus carthusianorum.

Dianthus carthusianorum is one of the dominant plant species colonising the Zn-Pb waste deposits in Boleslaw, Southern Poland. It differs in terms of morphology and genetics from ecotypes inhabiting non-metal-polluted areas. The response of waste-heap (metallicolous, M) and reference (nonmetallicolous, NM) ecotypes of D. carthusianorum to Pb in hydroponics was investigated and compared in this study. The plants of the M ecotype were more tolerant to Pb than these of the NM ecotype in spite of accumulation of higher concentrations of Pb. In both ecotypes, about 70-78% of Pb was retained in roots. In non Pb-treated plants, a higher glutathione (GSH) level was found in the M ecotype. After the Pb exposure, the GSH level decreased and was similar in both ecotypes. Lead treatment induced synthesis of phytochelatins (PCs) only in the plant roots, with significantly higher concentrations thereof detected in the NM ecotype. Malate and citrate concentrations were higher in the M ecotype; however, they did not change significantly upon any Pb treatment in either ecotype. The results indicated that neither PCs nor organic acids were responsible for the enhanced Pb tolerance of the waste-heap plants.

Effect of rotational shepherding on demographic and genetic connectivity of calcareous grassland plants.

Response to habitat fragmentation may not be generalized among species, in particular for plant communities with a variety of dispersal traits. Calcareous grasslands are one of the most species-rich habitats in Central Europe, but abandonment of traditional management has caused a dramatic decline of calcareous grassland species. In the Southern Franconian Alb in Germany, reintroduction of rotational shepherding in previously abandoned grasslands has restored species diversity, and it has been suggested that sheep support seed dispersal among grasslands. We tested the effect of rotational shepherding on demographic and genetic connectivity of calcareous grassland specialist plants and whether the response of plant populations to shepherding was limited to species dispersed by animals (zoochory). Specifically, we tested competing dispersal models and source and focal patch properties to explain landscape connectivity with patch-occupancy data of 31 species. We fitted the same connectivity models to patch occupancy and nuclear microsatellite data for the herb Dianthus carthusianorum (Carthusian pink). For 27 species, patch connectivity was explained by dispersal by rotational shepherding regardless of adaptations to zoochory, whereas population size (16% species) and patch area (0% species) of source patches were not important predictors of patch occupancy in most species. [Correction made after online publication, February 25, 2014: Population size and patch area percentages were mistakenly inverted, and have now been fixed.] Microsite diversity of focal patches significantly increased the model variance explained by patch occupancy in 90% of the species. For D. carthusianorum, patch connectivity through rotational shepherding explained both patch occupancy and population genetic diversity. Our results suggest shepherding provides dispersal for multiple plant species regardless of their dispersal adaptations and thus offers a useful approach to restore plant diversity in fragmented calcareous grasslands.

Flower colour and cytochromes P450.

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

Morphological, physiological, and genetic variation between metallicolous and nonmetallicolous populations of Dianthus carthusianorum.

Waste deposits produced by metal mining and smelting activities provide extremely difficult habitats for plant colonization and growth. Therefore, plants spontaneously colonizing such areas represent a very interesting system for studying evolution of plant adaptation and population differentiation between contaminated and noncontaminated environments. In this study, two populations of Dianthus carthusianorum, one originating from Zn-Pb waste deposit (a metallicolous population, M) and the other from unpolluted soil (a nonmetallicolous population, NM), were analyzed in respect of their morphological and physiological traits as well as genetic markers. It was found that the plants inhabiting the waste heap differed significantly from the NM plants in terms of leaf size and shape, and these differences were persistent between the first generation of the plants of both populations cultivated under uniform, controlled laboratory conditions. In contrast with the evident morphological differences, no significant differentiation between the populations regarding the physiological traits measured (accumulation of proline, anthocyanins, chlorophyll, carotenoids) was found. These traits can be regarded as neither population specific nor stress markers. The genetic variability was analyzed using 17 random amplified polymorphic DNA (RAPD) and four inter simple sequence repeat (ISSR) markers; this proved that the differentiation between the M and NM populations exists also at the genetic level. Analysis of molecular variance (AMOVA) showed that 24% of the total genetic diversity resided among populations, while 76% - within the populations. However, no significant differences in intrapopulation genetic diversity (Hj) between the M and NM populations of D. carthusianorum was found, which contradicts the theory that acquisition of adaptation mechanisms to adverse, isolated growth habitats is related to reduction in genetic diversity. Distinct genetic differences between the two populations in combination with evident morphological variation support the proposal to regard the M population of D. carthusianorum as a separate calamine ecotype.