Calcium-dependent protein kinase 16 phosphorylates and activates the aquaporin PIP2;2 to regulate reversible flower opening in Gentiana scabra.

Flower opening is important for successful pollination in many plant species, and some species repeatedly open and close their flowers. This is thought to be due to turgor pressure changes caused by water influx/efflux, which depends on osmotic oscillations in the cells. In some ornamental plants, water-transporting aquaporins, also known as plasma membrane intrinsic proteins (PIPs), may play an important role in flower opening. However, the molecular mechanism(s) involved in corolla movement are largely unknown. Gentian (Gentiana spp.) flowers undergo reversible movement in response to temperature and light stimuli; using gentian as a model, we showed that the Gentiana scabra aquaporins GsPIP2;2 and GsPIP2;7 regulate repeated flower opening. In particular, phosphorylation of a C-terminal serine residue of GsPIP2;2 is important for its transport activity and relates closely to the flower re-opening rate. Furthermore, GsPIP2;2 is phosphorylated and activated by the calcium (Ca2+)-dependent protein kinase GsCPK16, which is activated by elevated cytosolic Ca2+ levels in response to temperature and light stimuli. We propose that GsCPK16-dependent phosphorylation and activation of GsPIP2;2 regulate gentian flower re-opening, with stimulus-induced Ca2+ signals acting as triggers.

Genomic characterization of WRKY transcription factors related to secoiridoid biosynthesis in Gentiana macrophylla.

Gentiana macrophylla is one of Chinese herbal medicines in which 4 kinds of iridoids or secoiridoids, such as loganic acid, sweroside, swertiamarin, and gentiopicroside, are identified as the dominant medicinal secondary metabolites. WRKY, as a large family of transcription factors (TFs), plays an important role in the synthesis of secondary metabolites in plants. Therefore, WRKY genes involved in the biosynthesis of secoiridoids in G. macrophylla were systematically studied. First, a comprehensive genome-wide analysis was performed, and 42 GmWRKY genes were identified, which were unevenly distributed in 12 chromosomes. Accordingly, gene structure, collinearity, sequence alignment, phylogenetic, conserved motif and promoter analyses were performed, and the GmWRKY proteins were divided into three subfamilies based on phylogenetic and multiple sequence alignment analyses. Moreover, the enzyme-encoding genes of the secoiridoid biosynthesis pathway and their promoters were then analysed, and the contents of the four secoiridoids were determined in different tissues. Accordingly, correlation analysis was performed using Pearson's correlation coefficient to construct WRKY gene-enzyme-encoding genes and WRKY gene-metabolite networks. Meanwhile, G. macrophylla seedlings were treated with methyl jasmonate (MeJA) to detect the dynamic change trend of GmWRKYs, biosynthetic genes, and medicinal ingredient accumulation. Thus, a total of 12 GmWRKYs were identified to be involved in the biosynthesis of secoiridoids, of which 8 (GmWRKY1, 6, 12, 17, 33, 34, 38 and 39) were found to regulate the synthesis of gentiopicroside, and 4 (GmWRKY7, 14, 26 and 41) were found to regulate the synthesis of loganic acid. Taken together, this study systematically identified WRKY transcription factors related to the biosynthesis of secoiridoids in G. macrophylla, which could be used as a cue for further investigation of WRKY gene functions in secondary metabolite accumulation.

Transcriptome and metabolome analysis reveals mechanism of light intensity modulating iridoid biosynthesis in Gentiana macrophylla Pall.

Light intensity is a key factor affecting the synthesis of secondary metabolites in plants. However, the response mechanisms of metabolites and genes in Gentiana macrophylla under different light intensities have not been determined. In the present study, G. macrophylla seedlings were treated with LED light intensities of 15 µmol/m2/s (low light, LL), 90 µmol/m2/s (medium light, ML), and 200 µmol/m2/s (high light, HL), and leaves were collected on the 5th day for further investigation. A total of 2162 metabolites were detected, in which, the most abundant metabolites were identified as flavonoids, carbohydrates, terpenoids and amino acids. A total of 3313 and 613 differentially expressed genes (DEGs) were identified in the LL and HL groups compared with the ML group, respectively, mainly enriched in KEGG pathways such as carotenoid biosynthesis, carbon metabolism, glycolysis/gluconeogenesis, amino acids biosynthesis, plant MAPK pathway and plant hormone signaling. Besides, the transcription factors of GmMYB5 and GmbHLH20 were determined to be significantly correlated with loganic acid biosynthesis; the expression of photosystem-related enzyme genes was altered under different light intensities, regulating the expression of enzyme genes involved in the carotenoid, chlorophyll, glycolysis and amino acids pathway, then affecting their metabolic biosynthesis. As a result, low light inhibited photosynthesis, delayed glycolysis, thus, increased certain amino acids and decreased loganic acid production, while high light got an opposite trend. Our research contributed significantly to understand the molecular mechanism of light intensity in controlling metabolic accumulation in G. macrophylla.

Identification of a novel viral factor inducing tumorous symptoms by disturbing vascular development in planta.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development. IMPORTANCE While various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

Establishment of an Infection System for Gentian (Gentiana spp.) Sclerotial Flower Blight Disease.

Gentians (Gentiana spp.) as floriculture crops are constantly exposed to several fungal and viral pathogens in the field. Among the fungal diseases afflicting gentian production, gentian sclerotial flower blight caused by Ciborinia gentianae incurs economic losses, as it affects flowers before and after harvest. Currently, preventive measures for this disease are limited, and no resistant cultivars have been reported. This is partly because of the lack of a reliable infection system that could promote research on this plant-fungus interaction. In this study, Gentiana plant tissue culture material was inoculated with C. gentianae culture filtrate. We successfully demonstrated non-ascospore-mediated infection of C. gentianae. Inoculation of individual hyphal structures present in the culture filtrate suggested that sclerotial primordia are the main agents of this infection. Interestingly, our results indicated that primary infection of C. gentianae occurs in petals rather than leaves, which enables systemic infection and therefore mirrors the fungus's infection strategy observed in the field. Moreover, we showed that (i) non-ascospore hyphal structures can also cause disease in flowers grown in the field, and (ii) ascosporic infection can also be observed using the in vitro system, opening possibilities for both practical and basic research aimed to combat gentian sclerotial flower blight disease.

Response of selenium pools to drought stress by regulating physio­biochemical attributes and anatomical changes in Gentiana macrophylla.

Selenium (Se), as a vital stress ameliorant, possesses a beneficial effect on mediating detrimental effects of environmental threats. However, the mechanisms of Se in mitigating the deleterious effects of drought are still poorly understood. Gentiana macrophylla Pall. is a well-known Chinese medicinal herb, and its root, as the main medicinal site, has significant therapeutic effects. The purpose of this experiment was to investigate the functions of Se on the seedling growth and physiobiochemical characteristics in G. macrophylla subjected to drought stress. The changes in microstructure and chloroplast ultrastructure of G. macrophylla leaves under drought exposure were characterized by scanning electron microscopy (SEM), scanning electron microscopes and energy dispersive X-Ray spectroscope (SEM-EDX), and transmission electron microscopy (TEM), respectively. Results revealed that drought stress induced a notable increase in oxidative toxicity in G. macrophylla, as evidenced by elevated levels of hydrogen peroxide (H2O2), lipid peroxidation (MDA), enhanced antioxidative response, decreased plant photosynthetic function, and inhibited plant growth. Chloroplasts integrity with damaged membranes and excess osmiophilic granule were observed in the drought-stressed plants. Se supplementation notably recovered the stomatal morphology, anatomical structure damage, and chloroplast ultrastructure of G. macrophylla leaves caused by drought exposure. Exogenous Se application markedly enhanced SPAD, photosynthetic stomatal exchange parameters, and photosystem II activity. Se supplementation significantly promoted the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), while reducing levels of MDA, superoxide anion (O2-.) and H2O2, and improving membrane integrity. Furthermore, the ameliorative effects of Se were also suggested by increased contents of osmotic substances (soluble sugar and proline), boosted content of gentiopicroside and loganinic acid in roots, and alleviated the inhibition in plant growth and biomass. Fourier transform infrared (FTIR) analysis of Se-treated G. macrophylla roots under drought stress demonstrated that Se-stimulated metabolites including O-H, C-H, N-H, C-N, and CO functional groups, were involved in resisting drought stress. Correlation analysis indicated an obvious negative correlation between growth parameters and MDA, O2-. and H2O2 content, while a positive correlation with photosynthetic gas exchange parameters. Principal component analysis (PCA) results explained the total variance into two principal components contributing the maximum (93.50 %) among the drought exposure with or without Se due to the various experiment indexes. In conclusion, Se exerts beneficial properties on drought-induced detrimental effects in G. macrophylla by relieving oxidative stress, improving photosynthesis indexes, PSII activity, regulating anatomical changes, altering levels of gentiopicroside and loganinic acid, and promoting growth of drought-stressed G. macrophylla.

Gentiana capitata Buch.-Ham. ex D.Don Cell Suspension Culture as a New Source of Isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside and Their Effect on PC-12 Cell Viability.

Some species of the Gentianaceae family are a valuable source of secondary metabolites. However, the phytochemical knowledge of some of these species remains insufficient. Therefore, this work focused on the isolation of the two main secondary metabolites in the methanolic extract from a Gentiana capitata cell suspension using preparative HPLC and the determination of their structure using UHPLC-DAD-IT-MS/MS and NMR methods. Their content in the methanolic extract was quantified using a previously validated HPLC method. The toxicity of the extract and two isolated compounds was also tested on the PC-12 cell line. The structures of the main secondary metabolites were identified as isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside by comparing the UHPLC-DAD-IT-MS/MS and NMR results with the literature data. The content of isosaponarin was determined to be 0.76 ± 0.04%, and the content of 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside was found to be 0.31 ± 0.02% in the dry extract. Additionally, a two-fold increase in the viability of the PC-12 cell line was observed compared to the control after treatment with the methanolic extract at a concentration of 500 µg/mL. These results suggest the potential use of G. capitata cell suspension methanolic extract as a new source of isosaponarin and 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside, highlighting their lack of toxicity to the PC-12 (rat pheochromocytoma) cell line.

Evaluation of the Antidiabetic Potential of Xanthone-Rich Extracts from Gentiana dinarica and Gentiana utriculosa.

Despite the existence of various therapeutic approaches, diabetes mellitus and its complications have been an increasing burden of mortality and disability globally. Hence, it is necessary to evaluate the efficacy and safety of medicinal plants to support existing drugs in treating diabetes. Xanthones, the main secondary metabolites found in Gentiana dinarica and Gentiana utriculosa, display various biological activities. In in vitro cultured and particularly in genetically transformed G. dinarica and G. utriculosa roots, there is a higher content of xanthones. The aim of this study was to investigate and compare antidiabetic properties of secondary metabolites (extracts) prepared from these two Gentiana species, cultured in vitro and genetically transformed with those collected from nature. We compare HPLC secondary metabolite profiles and the content of the main extract compounds of G. dinarica and G. utriculosa methanol extracts with their ability to scavenge DPPH free radicals and inhibit intestinal α-glucosidase in vitro. Anti-hyperglycemic activity of selected extracts was tested further in vivo on glucose-loaded Wistar rats. Our findings reveal that the most prominent radical scavenging potential and potential to control the rise in glucose level, detected in xanthone-rich extracts, were in direct correlation with an accumulation of xanthones norswertianin and norswertianin-1-O-primeveroside in G. dinarica and decussatin and decussatin-1-O-primeveroside in G. utriculosa.

[A new benzopyran glycoside from Gentiana macrophylla].

Thirteen compounds were isolated and identified from 70% ethanol extract of the roots of Gentiana macrophylla by multi-chromatographic methods, including microporous resin, silica gel, and C_(18) reversed-phase column chromatography, as well as HPLC as follows: macrophylloside G(1), macrophylloside D(2), 5-formyl-2,3-dihydroisocoumarin(3),(+)-medicarpin(4),(+)-syringaresinol(5), liquiritigenin(6),(3R)-sativanone(7),(3R)-3'-O-methylviolanone(8), 4,2',4'-trihydroxychalcone(9), latifolin(10), gentioxepine(11), 6α-hydroxycyclonerolidol(12), and ethyl linoleate(13). Compound 1 was a new benzopyran glycoside. Compounds 4, 6-10, 12, and 13 were isolated for the first time from Gentiana plants. Compounds 1 and 2 showed promising hepatoprotective activity against D-GalN-induced AML12 cell damage at the concentration of 10 µmol·L~(-1), and compound 2 exhibited more significant activity than silybin at the same concentration.

Quantification of mangiferin in streptozotocin-induced diabetic rat plasma using UPLC-MS/MS: Pharmacokinetic assessment of Gentiana rhodantha extract after oral administration.

Mangiferin, a key bioactive constituent in Gentiana rhodantha, has a favorable impact on reducing blood sugar. A selective and sensitive UPLC MS/MS approach was developed for determining mangiferin in diabetic rats. Employing acetonitrile protein precipitation, chromatographic separation utilized a 2.1×50 mm, 3.5µm C18 column with a mobile phase of 0.1% formic acid aqueous and 5mM ammonium acetate (A, 45%) and acetonitrile (B, 55%) at a 0.5mL min-1 flow rate. Quantification, employing the multiple reaction monitoring (MRM) mode, focused on precursor-to-product ion transitions at m/z 447.1→271.1 for baicalin m/z and 421.0→301.0 for mangiferin. Calibration curves demonstrated linearity in the 1.00~100ng/mL range, with a lower quantification limit for rat plasma set at 1.00ng/mL. Inter- and intra-day accuracies spanned -9.1% to 8.5% and mangiferin mean recovery varied from 82.3% to 86.7%. The adeptly utilized UPLC-MS/MS approach facilitated the exploration of mangiferin pharmacokinetics in diabetic rats.

Comparison of plastid genomes and ITS of two sister species in Gentiana and a discussion on potential threats for the endangered species from hybridization.

BACKGROUND: Gentiana rigescens Franchet is an endangered medicinal herb from the family Gentianaceae with medicinal values. Gentiana cephalantha Franchet is a sister species to G. rigescens possessing similar morphology and wider distribution. To explore the phylogeny of the two species and reveal potential hybridization, we adopted next-generation sequencing technology to acquire their complete chloroplast genomes from sympatric and allopatric distributions, as along with Sanger sequencing to produce the nrDNA ITS sequences. RESULTS: The plastid genomes were highly similar between G. rigescens and G. cephalantha. The lengths of the genomes ranged from 146,795 to 147,001 bp in G. rigescens and from 146,856 to 147,016 bp in G. cephalantha. All genomes consisted of 116 genes, including 78 protein-coding genes, 30 tRNA genes, four rRNA genes and four pseudogenes. The total length of the ITS sequence was 626 bp, including six informative sites. Heterozygotes occurred intensively in individuals from sympatric distribution. Phylogenetic analysis was performed based on chloroplast genomes, coding sequences (CDS), hypervariable sequences (HVR), and nrDNA ITS. Analysis based on all the datasets showed that G. rigescens and G. cephalantha formed a monophyly. The two species were well separated in phylogenetic trees using ITS, except for potential hybrids, but were mixed based on plastid genomes. This study supports that G. rigescens and G. cephalantha are closely related, but independent species. However, hybridization was confirmed to occur frequently between G. rigescens and G. cephalantha in sympatric distribution owing to the lack of stable reproductive barriers. Asymmetric introgression, along with hybridization and backcrossing, may probably lead to genetic swamping and even extinction of G. rigescens. CONCLUSION: G. rigescens and G. cephalantha are recently diverged species which might not have undergone stable post-zygotic isolation. Though plastid genome shows obvious advantage in exploring phylogenetic relationships of some complicated genera, the intrinsic phylogeny was not revealed because of matrilineal inheritance here; nuclear genomes or regions are hence crucial for uncovering the truth. As an endangered species, G. rigescens faces serious threats from both natural hybridization and human activities; therefore, a balance between conservation and utilization of the species is extremely critical in formulating conservation strategies.

Comparative transcriptome analyses of three Gentiana species provides signals for the molecular footprints of selection effects and the phylogenetic relationships.

Species in Gentiana section Cruciata are important alpine plants with a center of diversity and speciation in Qinghai-Tibet Plateau (QTP), and some of these species are sympatrically distributed in northeastern QTP. Studies on genome features and natural selection signatures of sympatric species in section Crucata have been impeded by a lack of genomic resources. Here, we showed transcript characterizations and molecular footprints of selection effects on G. straminea, G. dahurica and G. officinalis based on the comparative transcriptome. A total of 62.97 Gb clean reads were obtained with unigene numbers per species ranging from 141,819 to 236,408 after assembly. We found that these three species had similar distribution of functional categories in different databases, and key enzyme-encoding genes involved in the iridoids biosynthesis were also obtained. The selective pressure analyses indicated that most paired orthologs between these three species were subject to negative selection, and only a low proportion of the orthologs that underwent positive selection were detected. We found that some positive selected genes were involved in "catalytic activity", "metabolic process", "response to stimulus" and "response to stress". Besides, large numbers of SSR primer pairs with transferabilities were successfully designed based on the available transcriptome datasets of three Gentiana species. The phylogenetic relationships reconstructed based on 352 single-copy nuclear genes provided a rough phylogenetic framework for this genus and confirmed the monophyly of section Cruciata. Our study not only provides insights for the natural selection effects on sympatric Gentiana species, but also enhances future genetic breeding or evolutionary studies on Qinjiao species.

Gentian FLOWERING LOCUS T orthologs regulate phase transitions: floral induction and endodormancy release.

Perennial plants undergo a dormant period in addition to the growth and flowering phases that are commonly observed in annuals and perennials. Consequently, the regulation of these phase transitions in perennials is believed to be complicated. Previous studies have proposed that orthologs of FLOWERING LOCUS T (FT) regulate not only floral initiation but also dormancy. We, therefore, investigated the involvement of FT orthologs (GtFT1 and GtFT2) during the phase transitions of the herbaceous perennial gentian (Gentiana triflora). Analysis of seasonal fluctuations in the expression of these genes revealed that GtFT1 expression increased prior to budbreak and flowering, whereas GtFT2 expression was induced by chilling temperatures with the highest expression occurring when endodormancy was released. The expression of FT-related transcription factors, reportedly involved in flowering, also fluctuated during each phase transition. These results suggested the involvement of GtFT1 in budbreak and floral induction and GtFT2 in dormancy regulation, implying that the two gentian FT orthologs activated a different set of transcription factors. Gentian ft2 mutants generated by CRISPR/Cas9-mediated genome editing had a lower frequency of budbreak and budbreak delay in overwintering buds caused by an incomplete endodormancy release. Our results highlighted that the gentian orthologs of FRUITFULL (GtFUL) and SHORT VEGETATIVE PHASE-like 1 (GtSVP-L1) act downstream of GtFT2, probably to prevent untimely budbreak during ecodormancy. These results suggest that each gentian FT ortholog regulates a different phase transition by having variable responses to endogenous or environmental cues, leading to their ability to induce the expression of distinct downstream genes.

Quality by design-based capillary electrophoresis method development for the quantitative analysis of four iridoid compounds in Gentiana macrophylla Radix.

In this study, the capillary electrophoresis-photodiode array detector was employed for the analysis of four iridoid compounds in Gentiana macrophylla Radix (RGM), and the method was optimized based on the concept of analytical quality by design (AQbD). The peak areas relative standard deviation (n = 3) and resolution of the four analytes were selected as critical method attributes. Fractional factorial design test combined with Pareto analysis were employed to screen critical method parameters (buffer concentration, pH, sodium dodecyl sulfate [SDS] micelle concentration, temperature, and voltage). Subsequently, three main factors (buffer concentration, buffer pH, and SDS concentration) were selected by central composite design test for constructing the design space. The optimal separation conditions as follows: capillary column (50.2 cm × 50 µm, detection length 40 cm). Working background electrolyte consisted of 51 mmol/L borax solution (pH = 9.47) and 40 mmol/L SDS. The samples were injected by pressure (5 s at 0.5 psi) and the detection was performed at 254 nm. Applied voltage was 20 kV and column temperature was 23°C. The developed method is rapid and reliable for the quantitative analysis of four iridoid compounds in RGM, providing a reference for the application of AQbD concept in the analysis of natural products.

The role of Gentiana lutea extracts in reducing UV-induced DNA damage.

Ultraviolet (UV) radiation can result in DNA damage, mainly through direct formation of pyrimidine dimers and generation of reactive oxygen species, which can lead to the skin disorders including cancer. In accordance with this, the use of natural antigenotoxins and/or antioxidants could contribute to human health protection. Considering that plants are rich in both, the aim of this study was to investigate UV-protective and antioxidative properties of yellow gentian (Gentiana lutea), being well established in pharmacopeias and traditional medicine. Tested extracts were derived from root and shoot of the in vitro cultivated plants. Prescreening of the genotoxic properties of UVC, UVA, and the extracts, as well as the extracts' antigenotoxicity were estimated by applying alkaline comet assay on normal fetal lung fibroblast (MRC-5) and human melanoma cells (Hs 294T). Antioxidant potential was tested in ferrous ions chelating ferric reducing antioxidant power and cupric reducing antioxidant capacity assays. Genotoxicity testing, which revealed moderate DNA-damaging potential of root extract on MRC-5 cells and high genotoxicity of shoot extract on both cell lines, pointed out nongenotoxic concentrations that could be used in antigenotoxicity assay. Doses of 63 and 3 J/cm2 for UVC and UVA, respectively, were established for antigenotoxicity study, since they induced sufficient DNA damage without notable cytotoxicity. Results of antigenotoxicity revealed strong protective effect of both extracts against UVC (the highest inhibitions 58% and 47%) and UVA (the highest inhibitions 69% and 60%), in Hs 294T and MRC-5 cells, respectively. Study of the antioxidative properties demonstrated stronger activity of shoot extract. Results obtained proved to be encouraging but further research of the UV-protective role of Gentiana lutea extracts and underlying molecular mechanisms is recommended.

Hybridization and divergent climatic preferences drive divergence of two allopatric Gentiana species on the Qinghai-Tibet Plateau.

BACKGROUND AND AIMS: Exploring how species diverge is vital for understanding the drivers of speciation. Factors such as geographical separation and ecological selection, hybridization, polyploidization and shifts in mating system are all major mechanisms of plant speciation, but their contributions to divergence are rarely well understood. Here we test these mechanisms in two plant species, Gentiana lhassica and G. hoae, with the goal of understanding recent allopatric species divergence on the Qinghai-Tibet Plateau (QTP). METHODS: We performed Bayesian clustering, phylogenetic analysis and estimates of hybridization using 561 302 nuclear genomic single nucleotide polymorphisms (SNPs). We performed redundancy analysis, and identified and annotated species-specific SNPs (ssSNPs) to explore the association between climatic preference and genetic divergence. We also estimated genome sizes using flow cytometry to test for overlooked polyploidy. KEY RESULTS: Genomic evidence confirms that G. lhassica and G. hoae are closely related but distinct species, while genome size estimates show divergence occurred without polyploidy. Gentiana hoae has significantly higher average FIS values than G. lhassica. Population clustering based on genomic SNPs shows no signature of recent hybridization, but each species is characterized by a distinct history of hybridization with congeners that has shaped genome-wide variation. Gentiana lhassica has captured the chloroplast and experienced introgression with a divergent gentian species, while G. hoae has experienced recurrent hybridization with related taxa. Species distribution modelling suggested range overlap in the Last Interglacial Period, while redundancy analysis showed that precipitation and temperature are the major climatic differences explaining the separation of the species. The species differ by 2993 ssSNPs, with genome annotation showing missense variants in genes involved in stress resistance. CONCLUSIONS: This study suggests that the distinctiveness of these species on the QTP is driven by a combination of hybridization, geographical isolation, mating system differences and evolution of divergent climatic preferences.

The impact of habitat loss on molecular signatures of coevolution between an iconic butterfly (Alcon blue) and its host plant (Marsh gentian).

Habitat loss is threatening natural communities worldwide. Small and isolated populations suffer from inbreeding and genetic drift, which jeopardize their long-term survival and adaptive capacities. However, the consequences of habitat loss for reciprocal coevolutionary interactions remain poorly studied. In this study, we investigated the effects of decreasing habitat patch size and connectivity associated with habitat loss on molecular signatures of coevolution in the Alcon blue butterfly (Phengaris alcon) and its most limited host, the marsh gentian (Gentiana pneumonanthe). Because reciprocal coevolution is characterized by negative frequency-dependent selection as a particular type of balancing selection, we investigated how signatures of balancing selection vary along a gradient of patch size and connectivity, using single nucleotide polymorphisms (SNPs). We found that signatures of coevolution were unaffected by patch characteristics in the host plants. On the other hand, more pronounced signatures of coevolution were observed in both spatially isolated and in large Alcon populations, together with pronounced spatial variation in SNPs that are putatively involved in coevolution. These findings suggest that habitat loss can facilitate coevolution in large butterfly populations through limiting swamping of locally beneficial alleles by maladaptive ones. We also found that allelic richness (Ar) of the coevolutionary SNPs is decoupled from neutral Ar in the butterfly, indicating that habitat loss has different effects on coevolutionary as compared with neutral processes. We conclude that this specialized coevolutionary system requires particular conservation interventions aiming at generating a spatial mosaic of both connected and of isolated habitat to maintain coevolutionary dynamics.

Impact of mycoheterotrophy on the growth of Gentiana zollingeri (Gentianaceae), as suggested by size variation, morphology, and 13C abundance of flowering shoots.

Gentiana zollingeri is an annual photosynthetic plant that employs a mycoheterotrophic growth strategy during its underground seedling stage (initial mycoheterotrophy). Notably, the morphological characteristics of its flowering shoots, such as shoot size, leaf size, and leaf color, are highly variable, and it was hypothesized that these variations may be linked to nutritional mode. The morphological characteristics of G. zollingeri individuals were thus investigated alongside environmental factors, 13C abundance, and diversity of colonizing arbuscular mycorrhizal (AM) fungi. The majority of G. zollingeri flowering individuals were found to exhibit a high affinity for the specific AM fungi that exclusively colonize roots of the mycoheterotrophic seedlings, while other phylogenetically diverse AM fungi could also be detected. The leaves to shoot dry weight ratio (leaf ratio) was negatively correlated with the canopy openness in the habitat, suggesting that leaf development is impeded in sunny conditions. Furthermore, the shoot weight of G. zollingeri was positively correlated with leaf 13C abundance. Given that 13C enrichment can provide indirect evidence of mycoheterotrophy in AM plants, the results suggest that the utilization of carbon obtained through mycoheterotrophy, at least during the underground seedling stage, is crucial for G. zollingeri.

The maleness of larger angiosperm flowers.

Flower biomass varies widely across the angiosperms. Each plant species invests a given amount of biomass to construct its sex organs. A comparative understanding of how this limited resource is partitioned among primary (male and female structures) and secondary (petals and sepals) sexual organs on hermaphrodite species can shed light on general evolutionary processes behind flower evolution. Here, we use allometries relating different flower biomass components across species to test the existence of broad allocation patterns across the angiosperms. Based on a global dataset with flower biomass spanning five orders of magnitude, we show that heavier angiosperm flowers tend to be male-biased and invest strongly in petals to promote pollen export, while lighter flowers tend to be female-biased and invest more in sepals to insure their own seed set. This result demonstrates that larger flowers are not simple carbon copies of small ones, indicating that sexual selection via male-male competition is an important driver of flower biomass evolution and sex allocation strategies across angiosperms.

Five New Glycoside Constituents from the Roots of Gentiana crassicaulis Duthie ex Burk.

Three undescribed glycoside constituents, macrophyllosides E-G and a pair of iridoid glycosides genticrasides A/B, together with eleven known glycoside compounds were isolated from the roots of Gentiana crassicaulis Duthie ex Burk. Their structures were identified by means of spectra analysis and data comparison with previous literatures. Interestingly, the glucose moieties in macrophylloside E and F possess free anomeric hydroxy groups. Genticrasides A/B, identified as a pair of iridoid originated lactones, have not been reported from Gentianaceae family up to now. The anti-inflammatory effects of selected compounds were also evaluated through the nitric oxide (NO) production inhibition in lipopolysaccharides (LPS)-induced RAW264.7 macrophage cells. In which, macrophyllosides G and D showed NO inhibitory activities with rates of 76.14±4.02 % and 52.44±8.29 % at 100 µg/mL.