Single molecule real-time sequencing data sets of Hypericum perforatum L. plantlets and cell suspension cultures.

Hypericum is a large genus that includes more than 500 species of pharmacological, ecological and conservation value. Although latest advances in sequencing technologies were extremely exploited for generating and assembling genomes of many living organisms, annotated whole genome sequence data is not publicly available for any of the Hypericum species so far. Bioavailability of secondary metabolites varies for different tissues and the data derived from different cultures will be a valuable tool for comparative studies. Here, we report the single molecule real-time sequencing (SMRT) data sets of Hypericum perforatum L. plantlets and cell suspension cultures for the first time. Sequencing data from cell suspension cultures yielded more than 33,000 high-quality transcripts from 20 Gb of raw data, while more than 55,000 high-quality transcripts were obtained from 35 Gb of raw data from plantlets. This dataset is a valuable tool for comparative transcriptomic analysis and will help to understand the unknown biosynthetic pathways of high medicinal value in the Hypericum genus.

Incidence and evolutionary relevance of autotriploid cytotypes in a relict member of the genus Daphne (Thymelaeaceae).

Odd ploidy-level cytotypes in sexually reproducing species are considered a dead end due to absent or reduced fertility. If sterility is only partial, however, their contribution to the population gene pool can be augmented by longevity and clonal growth. To test this, we investigated the cytotype origin and spatial pattern, and pollen viability in three relict shrub species of the genus Daphne (Thymelaeaceae Juss.) in central Europe. Daphne cneorum subsp. cneorum is a widespread European species that has a broad ecological amplitude, whereas D. cneorum subsp. arbusculoides and D. arbuscula are narrow endemics of the western Pannonian Plain and the Western Carpathians, respectively. Our study confirmed that all three taxa are diploid. However, of more than a thousand analysed individuals of D. cneorum subsp. cneorum, five in four different populations were triploid. Our data indicate that these triploids most likely originate from recurrent autopolyploidization events caused by the fusion of reduced and unreduced gametes. High pollen viability was observed in all three taxa and in both diploid and triploid cytotypes, ranging from 65 to 100 %. Our study highlights the significant role of odd ploidy-level cytotypes in interploidy gene flow, calling for more research into their reproduction, genetic variability, and overall fitness. Interestingly, while the endemic D. arbuscula differs from D. cneorum based on genetic and genome size data, D. cneorum subsp. arbusculoides was indistinguishable from D. cneorum subsp. cneorum. However, our study reveals that the subspecies differ in the number of flowers per inflorescence. This is the first comprehensive cytogeographic study of this intriguing genus at a regional scale, and in spite of its karyological stability, it contributes to our understanding of genomic evolution in plant species with a wide ecological amplitude.

Reproduction Modes and Conservation Implications in Three Polyploid Sorbus Stenoendemics in Eastern Slovakia (Central Europe).

The remarkable species diversity of the genus Sorbus is a result of polyploidization and frequent hybridization between interacting species of different cytotypes. Moreover, hybridization is possible between several parental taxa. Gametophytic apomixis, which is common among polyploid Sorbus taxa, indicates the role of clonal reproduction in the evolutionary stabilization of hybridogeneous genotypes. The precise determination of the origin of seeds and their quantitative evaluation may elucidate inter-cytotype interactions, the potential role of mixed-cytotype populations in evolutionary success, and the long-term survival of some hybrid species. We investigated the reproduction modes of selected species of Sorbus in mixed-cytotype populations in eastern Slovakia, Central Europe. We determined the pollen quality, seed production rate, and the ploidy level of mature trees, as well as the origin of the embryo and endosperm in seeds of the stenoendemics S. amici-petri, S. dolomiticola, and S. hornadensis. The tetraploids S. amici-petri and S. hornadensis are characterized by regular and highly stainable pollen grains and reproduce predominantly via pseudogamous apomixis. In contrast, triploid S. dolomiticola usually has oval, heterogenous, and weakly stainable pollen grains, suggesting male meiotic irregularities. Although seeds originate via pseudogamous apomixis in S. dolomiticola as well, the ploidy level of sperm cells participating in the fertilization of central cells is usually determined by co-occurring species of different cytotypes. This suggests that maintaining mating partners is necessary for the long-term survival of a triploid species. We documented rare BIII hybrids and the residual sexuality in tetraploids. The distribution of seeds of meiotic and apomeiotic origins in S. amici-petri shows bimodal characteristics; however, genotypes with predominantly sexual seed types are rare. Reproduction modes documented in polyploid stenoendemics of Sorbus and inferred microevolutionary intercytotype relationships highlight the mixed-cytotype populations as the source of biodiversity in apomictic plant complexes. We suggest that conservation efforts should focus on maintaining the species and cytotypic diversity of Sorbus populations, especially when it comes to the conservation of triploid species.

Variability of Reproduction Pathways in the Central-European Populations of Hawthorns with Emphasis on Triploids.

The role of apomeiosis, parthenogenesis, and pseudogamy in the asexual reproduction of some plant groups has not been fully elucidated in relation to species diversification. Quantitative analyses of seed origin may help in gaining better understanding of intercytotypic interactions. Asexual reproduction associated with polyploidy and frequent hybridization plays a crucial role in the evolutionary history of the genus Crataegus in North America. In Europe, the genus represents a taxonomically complex and very difficult species group not often studied using a modern biosystematic approach. We investigated the reproduction pathways in mixed-cytotype populations of selected taxa of Crataegus in eastern Slovakia, Central Europe. The investigated accessions were characterized by seed production data and the ploidy level of mature plants as well as the embryo and endosperm tissues of their seeds determined via flow cytometry. Diploid and polyploid hawthorns reproduce successfully; they also produce high numbers of seeds. An exception is represented by an almost sterile triploid. Diploids reproduce sexually. Polyploids shift to asexual reproduction, but pseudogamy seems to be essential for regular seed development. In rare cases, fertilization of unreduced gametes occurs, which offers opportunity for the establishment of new polyploid cytotypes between diploid sexuals and polyploid asexuals. Opposite to sexual diploids, triploids are obligate, and tetraploids almost obligate apomicts. Apomixis is considered to help stabilize individual weakly differentiated polyploid microspecies. Pseudogamy is a common feature and usually leads to unbalanced maternal to paternal contribution in the endosperm of triploid accessions. Parental contribution to endosperm gene dosage is somehow relaxed in triploids. Our Crataegus plant system resembles reproduction in the diploids and polyploids of North American hawthorns. Our data provide support for the hypothesis that polyploidization, shifts in reproduction modes, and hybridization shape the genus diversity also in Central Europe.

Genome size and endoreplication in two pairs of cytogenetically contrasting species of Pulmonaria (Boraginaceae) in Central Europe.

Genome size is species-specific feature and commonly constant in an organism. In various plants, DNA content in cell nucleus is commonly increased in process of endoreplication, cellular-specific multiplication of DNA content without mitosis. This leads to the endopolyploidy, the presence of multiplied chromosome sets in a subset of cells. The relationship of endopolyploidy to species-specific genome size is rarely analysed and is not fully understood. While negative correlation between genome size and endopolyploidy level is supposed, this is species- and lineage-specific. In the present study, we shed light on this topic, exploring both genome size and endoreplication-induced DNA content variation in two pairs of morphologically similar species of Pulmonaria, P. obscura-P. officinalis and P. mollis-P. murinii. We aim (i) to characterize genome size and chromosome numbers in these species using cytogenetic, root-tip squashing and flow cytometry (FCM) techniques; (ii) to investigate the degree of endopolyploidy in various plant organs, including the root, stem, leaf, calyx and corolla using FCM; and (iii) to comprehensively characterize and compare the level of endopolyploidy and DNA content in various organs of all four species in relation to species systematic relationships and genome size variation. We have confirmed the diploid-dysploid nature of chromosome complements, and divergent genome sizes for Pulmonaria species: P. murinii with 2n = 2x = 14, 2.31 pg/2C, P. obscura 2n = 2x = 14, 2.69 pg/2C, P. officinalis 2n = 2x = 16, 2.96 pg/2C and P. mollis 2n = 2x = 18, 3.18 pg/2C. Endopolyploidy varies between species and organs, and we have documented 4C-8C in all four organs and up to 32C (64C) endopolyploid nuclei in stems at least in some species. Two species with lower genome sizes tend to have higher endopolyploidy levels than their closest relatives. Endoreplication-generated tissue-specific mean DNA content is increased and more balanced among species in all four organs compared to genome size. Our results argue for the narrow relationship between genome size and endopolyploidy in the present plant group within the genus Pulmonaria, and endopolyploidization seems to play a compensatory developmental role in organs of related morphologically similar species.

Carex pulicaris abundance is positively associated with soil acidity, rainfall and floristic diversity in the eastern distribution range.

Carex pulicaris is considered an endangered species, and further losses are forecast under the influence of even moderate climate change. Local studies indicate that temporal declines in C. pulicaris abundance are positively correlated to decreases in precipitation and increases in air temperature. Determining ecological properties on larger scales than local ones can help develop effective protection programs for the species. We hypothesize that the local relationships observed between C. pulicaris abundance and precipitation, air temperature and soil properties will be confirmed in a spatially-oriented large-scale study performed in situ. Therefore, the present study takes a novel, large-scale integrated approach to (1) precisely characterize the ecological requirements of C. pulicaris within its eastern distribution range, and (2) determine the influence of its community type, soil properties and climatic conditions on its abundance. It was found that C. pulicaris is not a dominant or codominant species in the studied phytocoenoses in the eastern distribution range. Five natural vegetation groups including C. pulicaris, with significantly diverse species compositions, were resolved: well supported Estonian, Polish, Slovak and Radecz groups, and a weakly-supported Ambiguous group. The abundance of C. pulicaris was found to be positively correlated with the composition of the geographically-diversified plant communities and atmospheric precipitation, and to be also negatively associated with latitude and soil pH. Although the species is adapted to a relatively wide range of soil types, such adaptation requires appropriate substrate moisture level and light conditions. The species prefers moist organic and mineral soils and grows on both acid and neutral medium, characterized by a narrow C:N ratio, with various amounts of digestible total P, Mg and N, and low levels of digestible K. Climate change, manifested by reduced rainfall, may be one of the most important predictors negatively affecting the occurrence of C. pulicaris.

Phytochemical profiling of several Hypericum species identified using genetic markers.

In the present study, we performed phytochemical profiling of several under-exploited Hypericum representatives taxonomically belonging to the sections Ascyreia, Androsaemum, Inodora, Hypericum, Coridium, Myriandra, and Adenosepalum. The authenticity of the starting plant material was confirmed using the nuclear ribosomal internal transcribed spacer as a molecular marker, DNA content and chromosome number. Phenolic constituents were analyzed using high-performance liquid chromatography to complement species-specific metabolic profiles. In several Hypericum representatives, the pharmacologically important compounds, including naphthodianthrones; phloroglucinol derivatives; chlorogenic acid; and some classes of flavonoids, particularly the flavonols rutin and hyperoside, flavanol catechin, and flavanones naringenin and naringin, were reported for the first time. Comparative multivariate analysis of chemometric data for seedlings cultured in vitro and acclimated to the outdoor conditions revealed a strong genetically predetermined interspecific variability in phenolic compound content. In addition to hypericins, which are the most abundant chemomarkers for the genus Hypericum, rarely employed phenolic metabolites, including phloroglucinol derivatives, chlorogenic acid, catechin, naringenin, naringin, and kaempferol-3-O-glucoside, were shown to be useful for discriminating between closely related species. Given the increasing interest in natural products of the genus Hypericum, knowledge of the spectrum of phenolic compounds in shoot cultures is a prerequisite for future biotechnological applications. In addition, phytochemical profiling should be considered as an additional part of the integrated plant authentication system, which predominantly relies upon genetic markers.

Spatial and Temporal Patterns of Endopolyploidy in Mosses.

Somatic polyploidy or endopolyploidy is common in the plant kingdom; it ensures growth and allows adaptation to the environment. It is present in the majority of plant groups, including mosses. Endopolyploidy had only been previously studied in about 65 moss species, which represents less than 1% of known mosses. We analyzed 11 selected moss species to determine the spatial and temporal distribution of endopolyploidy using flow cytometry to identify patterns in ploidy levels among gametophytes and sporophytes. All of the studied mosses possessed cells with various ploidy levels in gametophytes, and four of six species investigated in sporophytic stage had endopolyploid sporophytes. The proportion of endopolyploid cells varied among organs, parts of gametophytes and sporophytes, and ontogenetic stages. Higher ploidy levels were seen in basal parts of gametophytes and sporophytes than in apical parts. Slight changes in ploidy levels were observed during ontogenesis in cultivated mosses; the youngest (apical) parts of thalli tend to have lower levels of endopolyploidy. Differences between parts of cauloid and phylloids of Plagiomnium ellipticum and Polytrichum formosum were also documented; proximal parts had higher levels of endopolyploidy than distal parts. Endopolyploidy is spatially and temporally differentiated in the gametophytes of endopolyploid mosses and follows a pattern similar to that seen in angiosperms.

Parasitic infection of Cobitis elongatoides Bacescu & Mayer, 1969 by zoonotic metacercariae Clinostomum complanatum (Rudolphi, 1814).

An infection of zoonotic Clinostomum complanatum metacercariae with potential human transmission was recorded close to fish farms in the Tisa River Basin of Slovakia and Ukraine. The prevalence varied from 19.4% to 81.3%, and the intensity of infection varied from 7 to 41. The results of a generalized linear model predicted a positive trend for the Cobitis elongatoides host and a standard length and intensity of infection, with females having a higher number of parasites. However, no significant impact was found of the intensity of infection on Clark's condition of the host. The metacercariae were primarily located in the anterior part of the host's body. Our study also showed significant evidence that water velocity affects the number of C. complanatum metacercariae, regardless of the host's (Cobitis) microhabitat.

Usnic acid, as a biotic factor, changes the ploidy level in mosses.

Lichens and mosses often share the same environmental conditions where they compete for substrate and other essential factors. Lichens use secondary metabolites as allelochemicals to repel surrounding plants and potential rivals. In mosses, endoreduplication leads to the occurrence of various ploidy levels in the same individual and has been suggested as an adaptation to abiotic stresses. Here, we show that also biotic factors such as usnic acid, an allelochemical produced by lichens, directly influenced the level of ploidy in mosses. Application of usnic acid changed the nuclei proportion and significantly enhanced the endoreduplication index in two moss species, Physcomitrella patens and Pohlia drummondii. These investigations add a new aspect on secondary metabolites of lichens which count as biotic factors and affect ploidy levels in mosses.

Identification of Onosma visianii Roots Extract and Purified Shikonin Derivatives as Potential Acaricidal Agents against Tetranychus urticae.

There is an increasing need for the discovery of reliable and eco-friendly pesticides and natural plant-derived products may play a crucial role as source of new active compounds. In this research, a lipophilic extract of Onosma visianii roots extract containing 12% of shikonin derivatives demonstrated significant toxicity and inhibition of oviposition against Tetranychus urticae mites. Extensive chromatographic separation allowed the isolation of 11 naphthoquinone derivatives that were identified by spectral techniques and were tested against Tetranychus urticae. All the isolated compounds presented effects against the considered mite and isobutylshikonin (1) and isovalerylshikonin (2) were the most active, being valuable model compounds for the study of new anti-mite agents.

Endoreduplication as a part of flower ontogeny in Trifolium pratense cultivars.

BACKGROUND: Endoreduplication appears in numerous plant species and plays a vital role during ontogeny. The presence of polyploid cells in an otherwise diploid organism is tied specifically to the taxonomy, ecology and physiology of the studied specimen. Little is known about the changes in endopolyploidy levels of floral organs during their development. In order to uncover the workings of endoreduplication in polysomatic species, our study examines flowers of T. pratense in three ontogenetic stages by means of flow cytometry. RESULTS: Cultivar 'Manuela' is characterized by the presence of 2C-8C and 'Dajana' 2C-16C nuclei. In general, the frequencies of nuclei only slightly changed during development. Endopolyploidy levels represented by endoreduplication index (EI) in the 'Manuela' sepals and stamens showed statistical differences between young and old stages, other organs of both cultivars between stages are not statistically different. Significant differences between 'Manuela' and 'Dajana' cultivars were found only in sepals of I. stage, and in petals and carpels of III. stage. Cultivars showed a similar pattern of endopolyploidy. However, a considerable decrease in EI 'Manuela' petals and carpels at III. stage was detected as opposed to 'Dajana'. Overall, a higher endoreduplication index is distinctive for organs of the 'Dajana' cultivar. CONCLUSIONS: In this study we prove the permanent presence of endopolyploid cells in the floral organs of T. pratense throughout their development.

Intra-population chemical polymorphism in Thymus pannonicus All. growing in Slovakia.

Solid-phase microextraction technique coupled with gas chromatography-flame ionisation detector and gas chromatography-mass spectrometry was used to investigate the inter- and intra-population chemical polymorphism in volatile constituents of three populations of Thymus pannonicus growing in Slovakia. To have an idea on what really the plant emits under different environmental and physiological conditions, a total of 86 individuals were separately analysed using low extraction temperatures (30°C). Data were analysed using a chemometric approach such as the principal component analysis which revealed a significant intra-population variability with the existence of up to four chemotypes: the most abundant is the p-cymene/γ-terpinene chemotype, followed by the geraniol and linalool chemotypes, and, only in one population, by the γ-muurolene/(E)-caryophyllene chemotype. This differentiation of individual plants inside the populations allows better adaptation to ecological conditions of localities and is probably connected with intrinsic genetic diversity of the species.