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Heavy metals can affect the morphology, physiology and evolution of plants. Asplenium viride is a diploid species, belonging to the largest genus of the cosmopolitan fern family Aspleniaceae, and occurring on various types of alkaline rocks. It is known to colonize sites with high concentrations of heavy metals, exhibiting changes in frond morphology. Microevolutionary processes, manifesting as changes in genome size and new genotype formation, can ultimately lead to the formation of new subspecies and speciation. This study aimed to evaluate the morphological and genetic diversity of A. viride, and to test for a potential correlation between variability and heavy metal concentration. Analysis of A. viride specimens, from one metalliferous site and five non-metalliferous localities, showed no apparent variation in genome size between plants from affected and non-affected sites. There was no significant correlation between genetic variability and heavy metal concentration. This was possibly due to intragametophytic selfing, caused by patchy habitats and subsequent founder effects, resulting from long-distance colonization by single spores.

Heavy metals can affect the morphology, physiology and evolution of plants. Asplenium viride is a diploid species, belonging to the largest genus of the cosmopolitan fern family Aspleniaceae, and occurring on various types of alkaline rocks. It is known to colonize sites with high concentrations of heavy metals, exhibiting changes in frond morphology. Microevolutionary processes, manifesting as changes in genome size and new genotype formation, can ultimately lead to the formation of new subspecies and speciation. This study aimed to evaluate the morphological and genetic diversity of A. viride, and to test for a potential correlation between variability and heavy metal concentration. Analysis of A. viride specimens, from one metalliferous site and five non-metalliferous localities, showed no apparent variation in genome size between plants from affected and non-affected sites. There was no significant correlation between genetic variability and heavy metal concentration. This was possibly due to intragametophytic selfing, caused by patchy habitats and subsequent founder effects, resulting from long-distance colonization by single spores.

Viola banksii of the eastern Australian and Tasmanian sect. Erpetion produces exclusively chasmogamous flowers, unlike most other temperate taxa of Viola which additionally produce obligatory self-pollinated cleistogamous flowers. This study explored flower structure and nyctinastic flower movements (temporal flower closure), the correlation of the timing of nyctinastic movements with stigma receptivity, and self-compatibility of flowers. Petal movement in day/night cycles was documented by two cameras. Floral morphology, anther protuberance anatomy, stigma receptivity, and pollen tube growth were examined using scanning electron microscopy, fluorescent microscopy, histochemical and sectioning techniques. Diurnal petal movements that cause flowers to be open during the day and closed during the night were documented in V. banksii. Chasmogamous flower colour, fragrance, anterior petal venation, and the indurated green area at the base of the anterior petal are all likely to play roles for pollinator attraction. Unlike most other Viola species, the anther protuberances do not function as nectaries. The flowers offer pollinators no nectar reward. The short time of stigma receptivity (2–3 days) of individual flowers limits opportunities for insect visitation and cross-pollination. Night-closed flowers of V. banksii appear to facilitate self-pollination. Self-compatibility was confirmed by tracking the pollen tube growth from the stigma to the ovule in spontaneous and hand self-pollinated flowers. Over the floral phenological cycle, nyctinasty coincides in time with stigma receptivity and, hence, the ability of self-pollination. We discovered that the same chasmogamous flower of V. banksii can cross-pollinate while open during the day, but self-pollinate while closed during the night (i.e. acting as a cleistogamous-like flower). Several floral characters indicate that V. banksii exhibits a pollination deceit strategy. Such a pollination system has not been described elsewhere in Viola.

Abstract The paper presents a technique for micropropagation of endangered in Europe and extinct in Poland Pulsatilla vulgaris for ex situ conservation of the genetic resources. Genotype-dependent induction of somatic embryogenesis and rooting was revealed in series of two experiments (I and II) performed under the same experimental conditions. Shoot tips of seedlings were the best explants in both experiments and Murashige and Skoog (MS) medium supplemented with 0.25 or 0.5 mg L−1 BAP was suitable for induction of somatic embryos (SE) and adventitious shoots. Mass SE was obtained in experiment I after explants transfer on ½ MS (2% sucrose) + 0.45 mg L−1 B1 and extending culture to 2–3 months without passages. Rooting of adventitious shoots was a critical point. Out of seven rooting media used in experiment I, only two, ½ MS hormone free (2% sucrose) + 0.45 mg L−1 B1 or MS + 5 mg L−1 NAA + 3.76 mg L−1 B2 resulted in altogether 36.4% rooted shoots. In experiment II, somatic embryogenesis, rooting and acclimatization of adventitious shoots failed. Regenerated plantlets and seedlings converted from SE from experiment I were acclimatized to ex vitro conditions. Both genome size, determined by flow cytometry, and genetic diversity analyzed by ISSR markers, confirmed the compatibility of regenerants from experiment I with P. vulgaris initial seedlings and commercial cultivar. Regenerants obtained in experiment II differed genetically from the regenerants of experiment I and cultivar. Propagated in vitro tissues/organs (SE, adventitious shoots) of P. vulgaris could be a source of material for cryopreservation, artificial seed production and/or for acclimatization of regenerated plantlets and could be used for restoration of the extinct populations. Key Message The micropropagation technique via organogenesis and somatic embryogenesis of endangered in Europe pasqueflower was developed as a tool for species recovery. The critical point is that somatic embryogenesis is genotype-dependent, which affects the repeatability of the experiments and also imposes applying molecular techniques to confirm the genetic fidelity of the regenerants with the initial material and other genotypes.

The Viola epipsila-V. palustris complex is a highly taxonomically complicated group of species in its entire circumboreal range of distribution. Habitat loss, forest flooding, and hybridization could lead to the extinction of V. epipsila. A hybrid index and principal component analysis (PCA) were used to select qualitative and quantitative morphological features to distinguish parent species and hybrids, inter simple sequence repeat (ISSR) markers to determine the genetic diversity of the populations, flow cytometry to estimate the genome size (GS), and non-coding chloroplast DNA (cpDNA) regions to indicate the directions of crosses. All taxa are very morphologically variable, and their features can change within a season. The most stable feature is the distance of the bracts on the pedicel from the rhizome. The genetic diversity of all taxa populations is low and highly influenced by selfing and vegetative propagation. The population structure is differentiated: populations of V. epipsila or V. palustris, mixed populations with both parent species, F1 hybrids and populations with introgressive forms occur in different regions. The interspecific GS variation corresponds to the ploidy level (4x = 2.52 pg, 8x = 4.26 pg, 6x = 3.42 pg). Viola epipsila is the mother plant of the hybrids. Research has shown the risk of V. epipsila extinction in Central Europe and the importance of local populations in studying the role of hybridization in reducing/maintaining/increasing biodiversity.

Viola arvensis cells were selected after treatment with Zn or Pb and regenerated into plants likely to have higher tolerance levels than the initial plant. The surviving cells in the suspension treated with 2000 μM of Zn, 2000 μM of Pb or 0 μM for 72 h were maintained on a solidified half-strength MS medium supplemented with 0.5 mg L−1 TDZ to induce divisions and organogenesis. The adventitious shoots obtained were rooted on a half-strength MS medium with 1 mg L−1 IBA. Regenerants derived from the Zn- and Pb-treated cells were vigorous and fully fertile. The in vitro conditions and metal impact generated a low genome alteration and overall low genetic diversity of regenerants compared to the initial plant and plants from the natural population. The cells of regenerants obtained after Pb treatment represented an approximately 12% higher tolerance level to Pb than the cells of the initial plant. This is the first report of plant regeneration from highly tolerant cells selected by heavy metal treatment. Regenerants successfully obtained in vitro could be considered as a source material for the recultivation of areas polluted with heavy metals.

In vitro regeneration of Viola stagnina Kit., endangered in most part of its European distribution range, was successfully obtained based on the newly developed protocol. Adventitious shoots via direct and indirect organogenesis were induced on leaf blade and petiole fragments on solidified Murashige and Skoog (MS) medium supplemented with 0.5 or 1 mg l−1 thidiazuron, respectively. Shoots were rooted on half-strength MS medium with 2% sucrose and 0.5 mg l−1 indole-3-acetic acid (IAA), and plantlets were successfully acclimatized. Sixty-five of the regenerated plants (72% of isolated shoots cultured on rooting medium) survived at the experimental plot conditions. Among recovered via organogenesis plants, individuals ‘true-to-type’ derived from initial plant G1 and also plants genetically distant from initial plants were detected by ISSR markers. In all groups of clones genetic indices (number of genotypes, polymorphic markers, gene diversity, total gene diversity, mean gene diversity) were lower than in natural populations. Regenerated plantlets had the same genome size estimated by flow cytometry as initial material and plants from natural populations. They developed chasmogamous flowers with highly viable pollen grains (over 90%), cleistogamous flowers, and set seeds from both flower types in the first and second seasons cultivated at experimental plots. This is the first report of a successfully developed micropropagation protocol of V. stagnina, and the first detailed genetic analysis of recovered plants with the use of ISSR markers and genome size measurements allowing to discuss the advantageous role of somaclonal variation in ex situ plant conservation with the use of in vitro micropropagation.

The use of cryopreservation techniques for plant species confined to wet habitats is difficult to standardize due to low tolerance for dehydration of plant tissue. The study aimed to develop a procedure for the long-term storage in liquid nitrogen (LN) of Viola stagnina, a rare and endangered species of moist habitats whose European range is constantly decreasing. Adventitious shoot apices of different sizes (small, medium and large) were cryopreserved using encapsulation/dehydration, encapsulation/vitrification (in PVS2 or PVS3 solutions) and droplet/vitrification methods. Encapsulation/dehydration was the most efficient technique, and 47.7 % regrowth was obtained with an average rate of shoot regeneration of 2.4 %. Less than 27.6 % of cryopreserved shoot apices were able to produce shoots with an efficiency of 0.35 or less based on vitrification procedures. In post-thaw recovery culture, adventitious shoots were produced indirectly (via callus). The genetic variation among recovered shoots after different cryopreservation methods was low (Dice coefficient not exceeded 0.09). Some clones were genetically uniform with the mother plant estimated by ISSR molecular markers. The genome size of all post-thaw recovered regenerants, except one individual with a multiplicated genome, was uniform with the initial plant. This paper presents the first report on cryopreservation of V. stagnina with a developed protocol that can be used in ex situ conservation of the species.

Viola reichenbachiana (2n = 4x = 20) and V. riviniana (2n = 8x = 40) are closely related species widely distributed in Europe, often sharing the same habitat throughout their overlapping ranges. It has been suggested in numerous studies that their high intraspecific morphological variability and plasticity might have been further increased by interspecific hybridisation in contact zones, given the sympatry of the species and the incomplete sterility of their hybrid. The aims of this study were to: (i) confirm that V. reichenbachiana and V. riviniana have one 4x genome in common, and (ii) determine the impact of hybridisation and introgression on genetic variation of these two species in selected European populations. For our study, we used 31 Viola populations from four European countries, which were analysed using AFLP and sequencing of a variable plastid intergenic spacer, trnH-psbA. Our analysis revealed that V. reichenbachiana exhibited larger haplotype diversity, having three species-specific haplotypes versus one in V. riviniana. The relationships among haplotypes suggest transfer of common haplotypes into V. riviniana from both V. reichenbachiana and hypothetically the other, now extinct, parental species. AFLP analysis showed low overall genetic diversity of both species, with V. riviniana showing higher among-population diversity. None of the morphologically designated hybrid populations had additive AFLP polymorphisms that would have indicated recent hybridisation. Also, kinship coefficients between both species did not indicate gene flow. V. riviniana showed significant population subdivision and significant isolation by distance, in contrast to V. reichenbachiana. The results indicate lack of gene flow between species, high influence of selfing on genetic variability, as well as probably only localised introgression toward V. riviniana.

Viola banksii, the type species of section Erpetion, is endemic in eastern mainland Australia. In this paper we characterise morphological and anatomical features and assess genome size and genetic diversity in combination with the breeding system. V. banksii develops exclusively chasmogamous flowers. Ovules are anatropous, crassinucellate and bitegmic, the female gametophyte is of the Polygonum type, and the embryo is of Asterad type surrounded by nuclear endosperm. Pollen is non-heteromorphic, 3-aperturate, and highly viable. V. banksii grows in shade on moist, well drained, often sandy soils, and this is reflected in the anatomy of its organs, which includes a lack of subepidermal collenchyma in aerial parts, large leaf epidermal cells with thin cell walls, a narrow cuticle layer, and vascular bundles with xylem that are not rich in vessels. V. banksii is tolerant to zinc and lead based on phytotoxicity test. The high chromosome number (2n = 10x = 50) does not correspond to a small genome size (2C DNA = 1.27 pg). Low mean intra-populational gene diversity (HS = 0.077) detected by ISSR markers confirms the strong influence of selfing and clonal propagation by pseudostolons. Unique morphological traits of V. banksii include nyctinastic petal movement, the lack of a floral spur, the presence of gland-like protuberances on two stamens, and the presence of pseudostolons, which could be a synapomorphy for the whole section.

Plants are exposed to various stress factors which might lead to structural damage and physiological function abnormalities. Drought is one of the environmental stress factors that reduce the productivity of plants. The aim of our study was to determine the influence of drought stress induced by mannitol (-0.5 and -1.5MPa) on selected physiological processes in Z. mays L. In the first stage we studied the effect of mannitol on the germination. In the second stage the effect of mannitol on the growth of plants germinated on distilled water and watered with mannitol in growth phase were measured. Mannitol, which decreased the water content in a concentration-dependent manner, had an inhibitory effect on germination and growth of seedlings and adult plants. Electrolyte leakage of cell membranes of the Z. mays seedlings showed high disturbances in the functioning of the membrane structures in the osmotic drought conditions. Similar results were obtained for maize roots, shoots and leaves in both treatment studies. Chlorophyll content showed only significant differences in plants from treated during the growth phase. Drought stress caused a decrease in chlorophyll content by almost a half compared to the control plants. Measurements of chlorophyll fluorescence of plant leaves from the second stage of experiments showed changes in fluorescence activity parameters Fv/Fm, NPQ, Rfd, qP, ect.; gas exchange measurements also showed changes in activity in each of the two phases.

Pollen and achene characters of natural interspecific hybrid Solidago ×niederederi Khek were analyzed and compared with putative parental species S. virgaurea L. and S. canadensis L. to estimate the level of disturbances in generative reproduction resulting from its hybrid nature. Pollen viability (stainability) of Solidago ×niederederi from one newly discovered locality in NE Poland was evidently reduced to ~65% in both viability tests (acetocarmine and Alexander). The diameter of viable pollen (median 21.11 µm) fell between S. canadensis (median 19.52 µm) and S. virgaurea (median 23.48 µm). Both parental species produced normally developed achenes with high frequency (~90%) whereas in the hybrid, the seed set was dramatically low (6%). The results clearly indicated that sexual reproduction of hybridogenous taxon S. ×niederederi is disturbed, and its potential impact as an invasive species depends mainly on vegetative propagation.

Environmental conditions, especially pollution of soils by heavy metals, can severely affect plant morphology, anatomy and reproduction. In present investigation, the morphological variability of forest violets ( Viola reichenbachiana and V. riviniana ), their degree of colonization by arbuscular mycorrhizal fungi (AMF) and their reproduction properties were studied using samples from ore-bearing areas with soils rich in heavy metals. In these habitats, individuals with typical morphological characters of V. reichenbachiana and V. riviniana were not identified but populations were intermediate between these two species. Three ore-bearing areas with soils of a high content but low extractable amounts of heavy metals (Pb, Zn, Cd) were covered by anthropogenic pine or beech forests, contrary to typical calamine heaps with metallophyte floras. Violets growing on ore-bearing areas were fully fertile, deposited Pb and Zn mainly in roots and had low Cd levels both in shoots and roots. Plants differed in AMF colonization with the degrees varying from nil to moderate. The violets growing in these areas were not affected in their reproduction and can be considered as stabilized introgressive forms or ecotypes of parental species on these soil conditions.

Viola uliginosa is an endangered species throughout its range in Central Europe. Based on an unusual combination of morphological characters, including a reported lack of cleistogamous (CL) flowers, it was previously placed in the monotypic subsection Repentes, section Viola. Available individual phylogenetic analyses identify V. uliginosa as a lineage deeply embedded in section Viola, but hitherto its exact position was unknown. Here we confirm the occurrence of cleistogamy in V. uliginosa. Unlike other species of section Viola, V. uliginosa produced both chasmogamous and CL flowers at the same time, not at different times of the year (seasonal cleistogamy). The two flower types differed conspicuously in morphology and microstructure, especially in size and shape of the pistil and stamens as confirmed by scanning electron microscopy. Secondly, we resolved the phylogenetic position of V. uliginosa as a lineage deeply embedded in subsection Rostratae by means of a multigene multispecies coalescent analysis using *BEAST. In this analysis, V. mirabilis and V. uliginosa were successive sisters to the rest of subsection Rostratae and, because allopolyploids exist between V. mirabilis and the last one, subsection Rostratae needs to comprise all these taxa to be monophyletic. The evolution of key morphological traits is briefly discussed.

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.

The genus ViolaL. with more than 500 species is difficult taxonomic group due to great intraspecific morphological variability resulted from the influence of environmental conditions but also from hybridization. Interspecific hybrids form hybrid swarm or introgressants, therefore species discrimination still remains a problem in this genus. To analyze intra- and interspecific variability the species from two sections were studied. Intraspecific (intra- and interpopulation) differentiation were analyzed in V. tricolorL. (section Melanium) from non-polluted and polluted with heavy metals sites. Intra- and interspecific variability with the influence of hybridization were studied in V. reichenbachiana Jord. ex Boreau – V. riviniana Rchb. group (section Viola). V. tricoloris a species very differentiated morphologically (intra- and interpopulation variability). It occupies metalliferous (Zn, Pb, Cd, Cu) and non-metalliferous sites in its geographic range. We analyzed morphological and anatomical features in metallicolous and non-metallicolous populations to determine the differences influenced by environmental conditions. In field observations, plants within both population types were quite variable, but interpopulation differences in some characters were evident (plant height, stem branching, number of flowers per plant, hairiness of whole plant and leaf, leaf shape and color, petal color, plant height, spur length, seed length). Correspondence analysis (CA) based on a data matrix of 12 selected qualitative and quantitative characters indicated that in terms of morphological variation the metallicolous populations did not possess common characters separating them from the non-metallicolous populations (Słomka et al. 2012). V. reichenbachianaand V. rivinianaare closely related species and due to a lack of prezygotic isolation mechanisms they hybridize in nature and easily produce interspecific hybrids and introgressive forms which occur sympatrically. Multivariate statistical analyses (MSA) based on 50 morphological characters of vegetative and generative organs showed significant differences between V. reichenbachianaand V. riviniana from non-polluted by heavy metals areas, high level of its intra- and interpopulational variability and the occurrence of interspecific hybrids. Plants from metal-polluted sites were very viable and formed a cluster of intermediate morphotypes between V. reichenbachianaand V. riviniana with some individuals included into V. riviniana range (Migdałek et al.2013, in press).

The plants growing in the natural environment are exposed to the influence of adverse factors which can cause metabolic disturbances, inhibition of growth, damage tissues and organs. The particular threats to plants are heavy metals, especially cadmium which inhibits plant growth and development. The aim of this study was to determine the influence of cadmium nitrate at concentrations of 0.01, 0.05 and 0.1 mM on seed germination, plants length, fresh and dry weight and the water content in Lupinus luteus L. and L. angustifolius L. The cadmium which is present in soil causes inhibition of seed germination, changes in root and hypocotyl length, value of fresh to dry weight ratio and the water content in plants organs.