Cardiac glycosides protect wormseed wallflower (Erysimum cheiranthoides) against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. Erysimum cheiranthoides (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model. We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in E. cheiranthoides and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using E. cheiranthoides cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field. EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side-chain cleavage, and EcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores, but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores. These results begin elucidation of the E. cheiranthoides cardiac glycoside biosynthetic pathway and demonstrate in vivo that cardiac glycoside production allows Erysimum to escape from some, but not all, specialist herbivores.

Hybridization and introgression are prevalent in Southern European Erysimum (Brassicaceae) species.

BACKGROUND AND AIMS: Hybridization is a common and important force in plant evolution. One of its outcomes is introgression - the transfer of small genomic regions from one taxon to another by hybridization and repeated backcrossing. This process is believed to be common in glacial refugia, where range expansions and contractions can lead to cycles of sympatry and isolation, creating conditions for extensive hybridization and introgression. Polyploidization is another genome-wide process with a major influence on plant evolution. Both hybridization and polyploidization can have complex effects on plant evolution. However, these effects are often difficult to understand in recently evolved species complexes. METHODS: We combined flow cytometry, analyses of transcriptomic sequences and pollen tube growth assays to investigate the consequences of polyploidization, hybridization and introgression on the recent evolution of several Erysimum (Brassicaceae) species from the South of the Iberian Peninsula, a well-known glacial refugium. This species complex differentiated in the last 2 million years, and its evolution has been hypothesized to be determined mainly by polyploidization, interspecific hybridization and introgression. KEY RESULTS: Our results support a scenario of widespread hybridization involving both extant and 'ghost' taxa. Several taxa studied here, most notably those with purple corollas, are polyploids, probably of allopolyploid origin. Moreover, hybridization in this group might be an ongoing phenomenon, as pre-zygotic barriers appeared weak in many cases. CONCLUSIONS: The evolution of Erysimum spp. has been determined by hybridization to a large extent. Species with purple (polyploids) and yellow flowers (mostly diploid) exhibit a strong signature of introgression in their genomes, indicating that hybridization occurred regardless of colour and across ploidy levels. Although the adaptive value of such genomic exchanges remains unclear, our results demonstrate the significance of hybridization for plant diversification, which should be taken into account when studying plant evolution.

BIOPHARMACEUTICAL UNDERSTANDING OF FORMULATION PREPARATION VARIABILITY OF PLGA NANOPARTICLES LOADED WITH ERYSIMUM EXTRACT.

The purpose of this study was to evaluate effect of process and formulation variables on the preparation of Erysimum extract loaded PLGA nanoparticles. The influence of the various biopharmaceutical factors such as type of organic solvent, type and concentration of surfactant, polymer concentration in the organic phase, ratio of organic phase and water phase were studied. Modified emulsification solvent evaporation method was used for preparation of nanoparticles. Based on the performed experiments optimal formulation of nanocomposite is suggested. Nanoparticle size, size distribution and entrapment efficiency were determined. Among five non-ionic surfactants polyvinyl alcohol provided more stable nanocomposite. Influence mechanisms of different surfactants on nanoparticle formation are provided. Water miscible organic solvent, acetone obtained 232 nm nanoparticles with improved size distribution. Entrapment efficiency was increased to 73% by reducing ratio of organic and water phases. Based on experiments nanoparticles with stable, reproducible properties are fabricated.

Less Is More: a Mutation in the Chemical Defense Pathway of Erysimum cheiranthoides (Brassicaceae) Reduces Total Cardenolide Abundance but Increases Resistance to Insect Herbivores.

Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hübner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.

Anther Rubbing, a New Mechanism That Actively Promotes Selfing in Plants.

Self-fertilization has recurrently evolved in plants, involving different strategies and traits and often loss of attractive functions, collectively known as the selfing syndrome. However, few traits that actively promote self-fertilization have been described. Here we describe a novel mechanism promoting self-fertilization in the Brassicaceae species Erysimum incanum. This mechanism, which we called "anther rubbing," consists of autonomous, repeated, and coordinated movements of the stamens over the stigma during flower opening. We have documented anther rubbing by time-lapse videos and experimentally show that it causes self-pollen deposition on stigmas and is sufficient to achieve maximal reproductive output in E. incanum. We predict that these movements should occur in species with limited inbreeding depression, and indeed we find that inbreeding depression in seed production is negligible in this species. While many studies have documented complex floral traits that promote outcrossing, the occurrence of anther rubbing demonstrates that plants can evolve elaborate and underappreciated adaptations to promote self-fertilization.

Pollination effectiveness in a generalist plant: adding the genetic component.

The pollination effectiveness of a flower visitor has traditionally been measured as the product of a quantity component that depends on the frequency of interaction and a quality component that measures the per-visit effects on plant reproduction. We propose that this could be complemented with a genetic component informing about each pollinator's contribution to the genetic diversity and composition of the plant progeny. We measured the quantity and quality components of effectiveness of most pollinator functional groups of the generalist herb Erysimum mediohispanicum. We used 10 microsatellite markers to calculate the genetic component as the diversity of sires among siblings and included it into the calculation of the pollination effectiveness. Functional groups varied in the quantity and quality components, which were shown to be decoupled. Functional groups also differed in the genetic component. This component changed the estimates of pollination effectiveness, increasing the differences between some functional groups and modifying the pollination effectiveness landscape. We demonstrate that including the genetic component in the calculation of the pollination effectiveness may allow a more complete quantification of the contribution of each pollinator to the reproductive success of a plant, providing information on its mating patterns and long-term fitness.

Efficacy of gummy lozenges containing Erysimum as ancillary treatment in children with sore throat.

BACKGROUND: Sore throat frequently occurs in children aged between four and fifteen years and is often associated to inflammation of the upper respiratory tract mucosa. A reliable approach to limit the damage caused by inflammation and, therefore, to alleviate associated symptoms might be the protection of the mucosa. Aim of this study was to assess the efficacy and tolerability of a medical device, formulated as a gummy lozenge and containing a combination of natural functional components (Erysimum, aloe vera and Xilogel®) able to exert a barrier effect on the mucosa, as ancillary treatment in children with sore throat. METHODS: This was an observational, prospective, parallel-group, multiple-dose trial of a medical device given in association to standard pharmacological prescribed therapy with an open label comparison vs. standard pharmacological prescribed therapy alone. The outcome measures of the study were assessed at baseline and after three days of treatment. RESULTS: One hundred and twelve school children with sore throat symptoms were recruited for this study and 69 were assigned to the group taking the study product. At the end of the treatment a statistically higher reduction in Sore Throat Pain Intensity Score and Pharyngitis Symptom Score was observed in the group taking the medical device. Moreover, the treatment with the medical device is associated to a statistically significant higher improvement of Child's General Conditions. The pediatrician assessed the efficacy and tolerability of the product under study as good/very good in 91% and 94%, respectively, of treated children. The consumer satisfaction questionnaire revealed that most of the children taking the lozenge rated it very positively in regard to its flavor and easiness of administration. CONCLUSIONS: The medical device used in this study may represent a valid choice as an adjuvant treatment in children with sore throat associated to upper respiratory tract infection.

Pollinator-mediated interactions in experimental arrays vary with neighbor identity.

PREMISE OF THE STUDY: Local ecological conditions influence the impact of species interactions on evolution and community structure. We investigated whether pollinator-mediated interactions between coflowering plants vary with plant density, coflowering neighbor identity, and flowering season. METHODS: We conducted a field experiment in which flowering time and floral neighborhood were manipulated in a factorial design. Early- and late-flowering Clarkia unguiculata plants were placed into arrays with C. biloba neighbors, noncongeneric neighbors, additional conspecific plants, or no additional plants as a density control. We compared whole-plant pollen limitation of seed set, pollinator behavior, and pollen deposition among treatments. KEY RESULTS: Interactions mediated by shared pollinators depended on the identity of the neighbor and possibly changed through time, although flowering-season comparisons were compromised by low early-season plant survival. Interactions with conspecific neighbors were likely competitive late in the season. Interactions with C. biloba appeared to involve facilitation or neutral interactions. Interactions with noncongeners were more consistently competitive. The community composition of pollinators varied among treatment combinations. CONCLUSIONS: Pollinator-mediated interactions involved competition and likely facilitation, depending on coflowering neighbor. Experimental manipulation helped to reveal context-dependent variation in indirect biotic interactions.

[STUDY OF CYTOTOXIC ACTIVITY OF METHANOL EXTRACTS OF THE ENDEMIC PLANT OF THE GENUS ERYSIMUM OF THE ADJARIAN FLORISTIC DISTRICT OF GEORGIA].

According to the results, investigated methanolic extracts of endemic plant from Adjarian floristic region of Georgia do not have specific cytotoxicity against the cell lines of breast adenocarcinoma MCF-7 human, which is typical, e.g., for plant pigment curcumin. At the same time, the high specific cytotoxicity of 2 and 3 fractions towards keratinocytes gives reason for considering endemic plant extracts, of Adjarian floristic region of Georgia, as a potential pharmacological means for topical treatment of pathologies caused by increased proliferation of keratinocytes, such as psoriasis.

Interaction of plant growth regulators and reactive oxygen species to regulate petal senescence in wallflowers (Erysimum linifolium).

BACKGROUND: In many species floral senescence is coordinated by ethylene. Endogenous levels rise, and exogenous application accelerates senescence. Furthermore, floral senescence is often associated with increased reactive oxygen species, and is delayed by exogenously applied cytokinin. However, how these processes are linked remains largely unresolved. Erysimum linifolium (wallflower) provides an excellent model for understanding these interactions due to its easily staged flowers and close taxonomic relationship to Arabidopsis. This has facilitated microarray analysis of gene expression during petal senescence and provided gene markers for following the effects of treatments on different regulatory pathways. RESULTS: In detached Erysimum linifolium (wallflower) flowers ethylene production peaks in open flowers. Furthermore senescence is delayed by treatments with the ethylene signalling inhibitor silver thiosulphate, and accelerated with ethylene released by 2-chloroethylphosphonic acid. Both treatments with exogenous cytokinin, or 6-methyl purine (which is an inhibitor of cytokinin oxidase), delay petal senescence. However, treatment with cytokinin also increases ethylene biosynthesis. Despite the similar effects on senescence, transcript abundance of gene markers is affected differentially by the treatments. A significant rise in transcript abundance of WLS73 (a putative aminocyclopropanecarboxylate oxidase) was abolished by cytokinin or 6-methyl purine treatments. In contrast, WFSAG12 transcript (a senescence marker) continued to accumulate significantly, albeit at a reduced rate. Silver thiosulphate suppressed the increase in transcript abundance both of WFSAG12 and WLS73. Activity of reactive oxygen species scavenging enzymes changed during senescence. Treatments that increased cytokinin levels, or inhibited ethylene action, reduced accumulation of hydrogen peroxide. Furthermore, although auxin levels rose with senescence, treatments that delayed early senescence did not affect transcript abundance of WPS46, an auxin-induced gene. CONCLUSIONS: A model for the interaction between cytokinins, ethylene, reactive oxygen species and auxin in the regulation of floral senescence in wallflowers is proposed. The combined increase in ethylene and reduction in cytokinin triggers the initiation of senescence and these two plant growth regulators directly or indirectly result in increased reactive oxygen species levels. A fall in conjugated auxin and/or the total auxin pool eventually triggers abscission.

Reproductive success through high pollinator visitation rates despite self incompatibility in an endangered wallflower.

PREMISE OF THE STUDY: Self incompatibility (SI) in rare plants presents a unique challenge-SI protects plants from inbreeding depression, but requires a sufficient number of mates and xenogamous pollination. Does SI persist in an endangered polyploid? Is pollinator visitation sufficient to ensure reproductive success? Is there evidence of inbreeding/outbreeding depression? We characterized the mating system, primary pollinators, pollen limitation, and inbreeding/outbreeding depression in Erysimum teretifolium to guide conservation efforts. METHODS: We compared seed production following self pollination and within- and between-population crosses. Pollen tubes were visualized after self pollinations and between-population pollinations. Pollen limitation was tested in the field. Pollinator observations were quantified using digital video. Inbreeding/outbreeding depression was assessed in progeny from self and outcross pollinations at early and later developmental stages. KEY RESULTS: Self-pollination reduced seed set by 6.5× and quadrupled reproductive failure compared with outcross pollination. Pollen tubes of some self pollinations were arrested at the stigmatic surface. Seed-set data indicated strong SI, and fruit-set data suggested partial SI. Pollinator diversity and visitation rates were high, and there was no evidence of pollen limitation. Inbreeding depression (δ) was weak for early developmental stages and strong for later developmental stages, with no evidence of outbreeding depression. CONCLUSIONS: The rare hexaploid E. teretifolium is largely self incompatible and suffers from late-acting inbreeding depression. Reproductive success in natural populations was accomplished through high pollinator visitation rates consistent with a lack of pollen limitation. Future reproductive health for this species will require large population sizes with sufficient mates and a robust pollinator community.

Evolution of pollination niches in a generalist plant clade.

It is widely assumed that floral diversification occurs by adaptive shifts between pollination niches. In contrast to specialized flowers, identifying pollination niches of generalist flowers is a challenge. Consequently, how generalist pollination niches evolve is largely unknown. We apply tools from network theory and comparative methods to investigate the evolution of pollination niches among generalist species belonging to the genus Erysimum. These species have similar flowers. We found that the studied species may be grouped in several multidimensional niches separated not by a shift of pollinators, but instead by quantitative variation in the relative abundance of pollinator functional groups. These pollination niches did not vary in generalization degree; we did not find any evolutionary trend toward specialization within the studied clade. Furthermore, the evolution of pollination niche fitted to a Brownian motion model without phylogenetic signal, and was characterized by frequent events of niche convergences and divergences. We presume that the evolution of Erysimum pollination niches has occurred mostly by recurrent shifts between slightly different generalized pollinator assemblages varying spatially as a mosaic and without any change in specialization degree. Most changes in pollination niches do not prompt floral divergence, a reason why adaptation to pollinators is uncommon in generalist plants.

Possible allostery and oligomerization of recombinant plastidial sn-glycerol-3-phosphate acyltransferase.

Plastidial acyl-acyl carrier protein:sn-glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the acyl-acyl carrier protein-dependent sn-1 acylation of sn-glycerol 3-phosphate (G3P) to produce lysophosphatic acid. Functional recombinant Erysimum asperum GPAT (EaGPAT), devoid of transit peptide, was produced in yeast. Analysis of the dependence of EaGPAT activity on increasing G3P concentration resulted in a hyperbolic response. EaGPAT exhibited a preference for 18-carbon unsaturated acyl-CoAs. Assays with concentrations of oleoyl-CoA up to 90µM revealed an exponential response to increasing concentrations of acyl donor, and the introduction of increasing concentrations of unlabeled linoleoyl-CoA into the standard reaction mixture resulted in increased incorporation of radiolabeled oleoyl moieties into lysophosphatidic acid. Collectively, the kinetic results suggest that acyl-CoA may act as both substrate and allosteric effector. EaGPAT was also shown to oligomerize to form higher molecular mass multimers, with the monomer and trimer being the predominant forms of the enzyme. Since most allosteric enzyme exhibit quaternary structure, the self-associating properties of EaGPAT are consistent with those of an allosteric enzyme. These results could have important regulatory implications when plastidial GPAT is introduced into a cytoplasmic environment where acyl-CoA is the acyl donor supporting cytoplasmic glycerolipid assembly.

Association between inbreeding depression and floral traits in a generalist-pollinated plant.

Individual variation in the magnitude of inbreeding depression (ID) in plants and its association with phenotypic traits may have important consequences for mating system evolution. This association has been investigated only scarcely, and always considering traits functionally related to autogamy. Here, we explore the association between individual variation in ID and plant traits associated with pollinator attractiveness (related to plant size, corolla size and corolla shape) in two populations of Erysimum mediohispanicum (Brassicaceae). ID was calculated along the entire life cycle of the plants. In addition, we also explored the relationship between phenotypic traits and the individual levels of heterozygosity. We found significant associations between ID and corolla diameter and stalk height, being taller plants with larger corollas those undergoing a lower intensity of ID. Furthermore, we found a negative relationship between corolla diameter and heterozygosity, suggesting that plants with large flowers have purged their genetic load. Finally, we found a significant effect of corolla diameter on the intrapopulation genetic structure. All these findings suggest that plants with large flowers have secularly suffered frequent inbreeding in the study populations. Because corolla diameter is a trait frequently selected by pollinators in E. mediohispanicum, we believe that the observed relationship between this trait and ID could be mediated by pollinators, probably throughout an increasing in biparental inbreeding, geitonogamy or autogamy.

Evolution of pollination niches and floral divergence in the generalist plant Erysimum mediohispanicum.

BACKGROUND AND AIMS: How generalist plants diverge in response to pollinator selection without becoming specialized is still unknown. This study explores this question, focusing on the evolution of the pollination system in the pollination generalist Erysimum mediohispanicum (Brassicaceae). METHODS: Pollinator assemblages were surveyed from 2001 to 2010 in 48 geo-referenced populations covering the entire geographic distribution of E. mediohispanicum. Bipartite modularity, a complex network tool, was used to find the pollination niche of each population. Evolution of the pollination niches and the correlated evolution of floral traits and pollination niches were explored using within-species comparative analyses. KEY RESULTS: Despite being generalists, the E. mediohispanicum populations studied can be classified into five pollination niches. The boundaries between niches were not sharp, the niches differing among them in the relative frequencies of the floral visitor functional groups. The absence of spatial autocorrelation and phylogenetic signal indicates that the niches were distributed in a phylogeographic mosaic. The ancestral E. mediohispanicum populations presumably belonged to the niche defined by a high number of beetle and ant visits. A correlated evolution was found between pollination niches and some floral traits, suggesting the existence of generalist pollination ecotypes. CONCLUSIONS: It is conjectured that the geographic variation in pollination niches has contributed to the observed floral divergence in E. mediohispanicum. The process mediating this floral divergence presumably has been adaptive wandering, but the adaptation to the local pollinator faunas has been not universal. The outcome is a landscape where a few populations locally adapted to their pollination environment (generalist pollination ecotypes) coexist with many populations where this local adaptation has failed and where the plant phenotype is not primarily shaped by pollinators.

Pre-copulatory choices drive post-copulatory decisions: mechanisms of female control shift across different life stages.

BACKGROUND: The 'wallflower' hypothesis proposes females mate indiscriminately to avoid reproductive delays. Post-copulatory mechanisms may then allow 'trading up', favouring paternity of future mates. We tested links between pre- and post-copulatory choice in Latrodectus geometricus female spiders paired sequentially with two males. These females copulate as adults or as subadults and store sperm in paired spermathecae. Choosy adults have a higher risk of delays to reproduction than subadults. RESULTS: We predicted low pre-copulatory, but high post-copulatory choice at first matings for adults and the opposite for subadults. At second matings, we expected all females would prefer males superior to their first. We found all females mated indiscriminately at their first pairing, but in contrast to subadults, adults usually allowed only a single insertion (leaving one of their paired spermatheca empty); a mechanism of post-copulatory choosiness. Adult-mated females were more likely to remate than subadult-mated females when they became adults, showing a preference for larger males, while subadult-mated females tended to prefer males of greater size-corrected mass. CONCLUSIONS: Our results show that the 'wallflower' effect and 'trading up' tactics can be utilized at different life stages, allowing females to employ choice even if rejecting males is costly.

Fitness consequences of centrality in mutualistic individual-based networks.

The relationships among the members of a population can be visualized using individual networks, where each individual is a node connected to each other by means of links describing the interactions. The centrality of a given node captures its importance within the network. We hypothesize that in mutualistic networks, the centrality of a node should benefit its fitness. We test this idea studying eight individual-based networks originated from the interaction between Erysimum mediohispanicum and its flower visitors. In these networks, each plant was considered a node and was connected to conspecifics sharing flower visitors. Centrality indicates how well connected is a given E. mediohispanicum individual with the rest of the co-occurring conspecifics because of sharing flower visitors. The centrality was estimated by three network metrics: betweenness, closeness and degree. The complex relationship between centrality, phenotype and fitness was explored by structural equation modelling. We found that the centrality of a plant was related to its fitness, with plants occupying central positions having higher fitness than those occupying peripheral positions. The structural equation models (SEMs) indicated that the centrality effect on fitness was not merely an effect of the abundance of visits and the species richness of visitors. Centrality has an effect even when simultaneously accounting for these predictors. The SEMs also indicated that the centrality effect on fitness was because of the specific phenotype of each plant, with attractive plants occupying central positions in networks, in relation to the distribution of conspecific phenotypes. This finding suggests that centrality, owing to its dependence on social interactions, may be an appropriate surrogate for the interacting phenotype of individuals.

Geometric morphometrics of corolla shape: dissecting components of symmetric and asymmetric variation in Erysimum mediohispanicum (Brassicaceae).

Symmetry is an important feature of floral structure, and floral symmetries are diverse and often complex. We use a new morphometric approach for analysing shapes with complex types of symmetry, which partitions shape variation into a component of symmetric variation and different components of asymmetry. This approach, based on the mathematical theory of symmetry groups, can be used for landmark configurations with any type of symmetry and is therefore promising as a general framework for morphometric analyses of floral symmetry and asymmetry. We demonstrate this approach by quantifying floral shape variation in a wild population of Erysimum mediohispanicum (Brassicaceae). Flowers of this species are disymmetric, so that the symmetry in the left-right and adaxial-abaxial directions can be considered separately and in combination. Both principal component analysis and Procrustes ANOVA indicate that symmetric variation accounts for most of the total variance and that adaxial-abaxial asymmetry is the dominant component of fluctuating asymmetry. Each component is associated with specific patterns of shape variation. These results illustrate the potential of the new method and suggest new areas for future research. The new morphometric approach is promising for further analyses of floral symmetry and asymmetry in evolutionary and developmental contexts.

Pollen limitation in a narrow endemic plant: geographical variation and driving factors.

Pollen limitation may have important consequences for the reproduction and abundance of plant species. It may be especially harmful to endangered and endemic plants with small populations. In this study, we quantify the effect of pollen limitation on seed production and seedling emergence in an endangered narrow endemic crucifer, Erysimum popovii. We conducted a pollen addition experiment across the entire geographic distribution of the species, and explored the effect of pollinator assemblage, plant population size and density, and other habitat variables on pollen limitation intensity in 13 populations. We supplemented flowers in 20 plants per population with allogamous pollen. To account for potential resource reallocation, we used two types of control untreated flowers: internal control flowers from the same individual as the supplemented flowers, and external control flowers from other individuals. Our results indicate that E. popovii is pollen-limited in most of the populations studied, but only through seed production, since pollen supplementation did not enhance seedling emergence. Beefly abundance was associated with among-population differences in pollen limitation intensity. Populations in which beeflies were more abundant were less pollen-limited. In contrast, the abundance of other flower visitors, such as large bees or butterflies, was not associated with pollen limitation. Annual rainfall and bare soil cover were associated with the intensity of pollen limitation across populations.

Mathematical modeling and optimizing the in vitro shoot proliferation of wallflower using multilayer perceptron non-dominated sorting genetic algorithm-II (MLP-NSGAII).

Novel computational methods such as artificial neural networks (ANNs) can facilitate modeling and predicting results of tissue culture experiments and thereby decrease the number of experimental treatments and combinations. The objective of the current study is modeling and predicting in vitro shoot proliferation of Erysimum cheiri (L.) Crantz, which is an important bedding flower and medicinal plant. Its micropropagation has not been investigated before and as a case study multilayer perceptron- non-dominated sorting genetic algorithm-II (MLP-NSGAII) can be applied. MLP was used for modeling three outputs including shoots number (SN), shoots length (SL), and callus weight (CW) based on four variables including 6-benzylaminopurine (BAP), kinetin (Kin), 1-naphthalene acetic acid (NAA) and gibberellic acid (GA3). The R2 correlation values of 0.84, 0.99 and 0.93 between experimental and predicted data were obtained for SN, SL, and CW, respectively. These results proved the high accuracy of MLP model. Afterwards the model connected to Non-dominated Sorting Genetic Algorithm-II (NSGA-II) was used to optimize input variables for obtaining the best predicted outputs. The results of sensitivity analysis indicated that SN and CW were more sensitive to BA, followed by Kin, NAA and GA. For SL, more sensitivity was obtained for GA3 than NAA. The validation experiment indicated that the difference between the validation data and MLP-NSGAII predicted data were negligible. Generally, MLP-NSGAII can be considered as a powerful method for modeling and optimizing in vitro studies.