Genetic Structure of Cochliobolus sativus Populations Sampled from Roots and Leaves of Barley and Wheat in North Dakota

Common root rot (CRR) and spot blotch, caused by Cochliobolus sativus (Ito and Kurib.) Drechsl. ex Dast., are important diseases of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) worldwide. However, the population biology of C. sativus is still poorly understood. In this study, the genetic structure of three C. sativus populations, consisting of isolates sampled respectively from barley leaves (BL), barley roots (BR) and wheat roots (WR) in North Dakota, was analysed with amplified fragment length polymorphism (AFLP) markers. A total of 127 AFLP loci were generated among 208 C. sativus isolates analysed with three primer combinations. Gene diversity (H = 0.277–0.335) were high in all three populations. Genetic variation among C. sativus individuals within population accounted for 74%, whereas 26% of the genetic variation was explained among populations. Genetic differentiation was high (ØPT = 0.261, corrected = 0.39), whereas gene flow (Nm) ranged from 1.27 to 1.56 among the three populations analysed. The multilocus linkage disequilibrium (LD) (= 0.076–0.117) was moderate in C  sativus populations. Cluster analyses indicate that C. sativus populations differentiated according to the hosts (barley and wheat) and tissues (root and leaf) although generalists also exist in North Dakota. Crop breeding may benefit from combining genes for resistance against both specialists and generalists of C. sativus.     

Effects of Epoxiconozale and Pyraclostrobin Fungicides in the Infection Process of Hemileia vastatrix on Coffee Leaves as Determined by Chlorophyll a Fluorescence Imaging

Coffee is the most traded commodity in the world, and Brazil is its largest producer. Coffee leaf rust, caused by the biotrophic fungus Hemileia vastatrix, is the most important coffee disease, reducing coffee yield by 35–50%. This study aimed to use the ratio of variable and maximum fluorescence of dark‐adapted tissue (F_v/F_m) as a parameter to differentiate presymptomatic tissue from healthy tissue during disease development in plants sprayed with pyraclostrobin and epoxiconazole after 4 days postinoculation. Visual severity was considered as an indicative of apparent disease and true severity as an indicative of both apparent and non‐apparent disease. There was a significant linear relationship between the areas of true severity and visual severity, and for each additional unit in the visual severity, there was an increase of 1.53 units on the true severity. For the epoxiconazole and pyraclostrobin treatments, coffee leaf rust symptoms decreased according to both visual and F_v/F_m images. Pustules on the leaves sprayed with epoxiconazole were smaller in size than those on the leaves of non‐sprayed plants but bigger than those sprayed with pyraclostrobin. The reduction in F_v/F_m values at the pustule epicentres present on the leaves of plants sprayed with epoxiconazole, and pyraclostrobin was greater than those of the non‐sprayed plants. This finding was expected and reflects the importance of these fungicides in prohibiting the progress of coffee leaf rust. The photosynthetic capacity of Coffea arabica was affected by H. vastatrix infection, and the F_v/F_m parameter was able to show this effect before the visual symptoms were noticed.     

Sensitivity of Zymoseptoria tritici Isolates from Tunisia to Pyraclostrobin,Fluxapyroxad, Epoxiconazole,Metconazole, Prochloraz and Tebuconazole

Sensitivity of 159 isolates of Zymoseptoria tritici collected from durum wheat fields in Tunisia in 2012 was analysed towards pyraclostrobin, fluxapyroxad, epoxiconazole, metconazole, prochloraz and tebuconazole using microtiter tests. All isolates were found to be highly sensitive to pyraclostrobin with EC₅₀ <0.01 mg/l with the exception of three isolates from the same field with higher EC₅₀ values (>0.5 mg/l). These three isolates carried a mutation in the cytochrome b gene encoding the G143A substitution. This is the first report of quinone outside inhibitors (QoI) resistance in Z. tritici in Tunisia. Sensitivity towards r fluxapyroxad was in a narrow range with EC₅₀ values ranging between 0.013 and 0.125 mg/l, which can serve as baseline sensitivity data for the future. Demethylation inhibitors sensitivity varied across a broad range with the data indicating a slight shift in sensitivity when compared to a previous study on the 2010 population. No highly sensitive strains were isolated from samples from fields, which had received three or four DMI applications.     

Chitosan combined with sodium silicate treatment induces resistance against rot caused by Alternaria alternata in postharvest jujube fruit

The effect of chitosan (0.1 mol/L) combined with sodium silicate (100 mmol/L) treatment on Alternaria rot caused by Alternaria alternata in postharvest jujube fruit (Ziziphus jujuba Mill. cv. Dongzao) was studied. The results showed that chitosan combined with sodium silicate treatment significantly reduced the lesion diameter, decay incidence, red index and weight loss of jujube fruit compared with control samples. Combining treatment increased the ascorbic acid, flavonoids, total phenolic compounds and lignin content. The level of superoxide anion () and hydrogen peroxide of treated samples was also increased compared with the control samples. Meanwhile, the activities of phenylalanine ammonia lyase, polyphenol oxidase, superoxide dismutase, peroxidase, chitinase and β‐1,3‐glucanase were also accumulated in treated jujube samples, while the activity of catalase markedly decreased. These results indicated that chitosan combined with sodium silicate treatment could induce the disease resistance of postharvest jujube. Therefore, coating postharvest jujube using chitosan combined with sodium silicate could promise as a novel method for preventing the disease infection of postharvest jujube.     

Variability of the Ramularia collo‐cygni Population in Central Europe

The genetic structure of the fungal barley pathogen Ramularia collo‐cygni (Rcc) population in Central Europe involving the isolates from the Czech Republic, the Slovak Republic, Germany and Swiss was determined using amplified fragment length polymorphism (AFLP) analysis. One hundred and eighty‐four markers were chosen to determine genetic and genotypic diversity and to test the hypothesis of random mating and population differentiation of Rcc isolates. Among the 337 isolates collected, the overall gene diversity was moderate ( = 0.216). The level of multilocus genotypic diversity was higher within populations than among them. All individuals had unique multilocus genotypes. Genetic differentiation was significant among populations in localities, but at a moderate level (θ = 0.12; P < 0.001), suggesting that gene flow is occurring among populations. The isolates from all twelve clusters produced by Structure were found in all local populations, although at different frequencies. Therefore, the inferred clusters did not represent geographical populations. Although the null hypothesis of random mating in Rcc populations was rejected, the high level of genotypic diversity suggests that the Rcc population structure appears to be generated by a mixed reproductive system including both asexual and sexual reproduction, along with a rather high migration rate.     

A Comparison of Different Estimation Methods for Fungicide EC50 and EC95 Values

EC₅₀ and EC₉₅ (the effective concentrations to cause inhibitions by 50 and 95%, respectively) are commonly used to express fungicide potency. Different methods are currently employed to calculate EC₅₀ and EC₉₅ values. In this study, EC₅₀ and EC₉₅ values for fungicide epoxiconazole against 34 isolates of Sclerotinia sclerotiorum were calculated with seven different methods. Results showed that for both EC₅₀ and EC₉₅ calculations, there was no significant difference among three statistical programs IBM spss ^®, GraphPad Prism^® and dps ^® (P ≥ 0.066). Methods linear log (linear regression of mycelial growth inhibition vs. logarithmic concentration) and interpolation log (linear interpolation from inhibition and logarithmic concentration data) were not significantly different (P ≥ 0.058) from IBM spss in EC₅₀ calculations. These results indicate that among the seven methods, the three statistical programs IBM spss , GraphPad Prism, dps and linear log method are appropriate for EC₅₀ calculations. But for EC₉₅ calculations, only the three statistical programs are recommended, and GraphPad Prism is likely to give a little higher values than spss and dps .     

Systemic fungicides reduce the abundance of yeast-like symbiotes and the performance of the planthopper Delphacodes kuscheli (Hemiptera: Delphacidae)

Delphacodes kuscheli is the main vector of Mal de Río Cuarto, the most important viral disease of maize in Argentina. This planthopper harbours obligate fungal mutualists, known as yeast-like symbiotes (YLS), which play a key role in the host life cycle. Here, we evaluated the efficacy of six systemic fungicides, prothioconazole (P), prothioconazole & trifloxystrobin (PT), pyraclostrobin & epoxiconazole (PE), pyraclostrobin, epoxiconazole & fluxapyroxad (PEF), picoxystrobin & ciproconazole (PC), and tebuconazole (T) on the reduction of the abundance of YLS in nymphs and newly emerged adults, and the impact of such reduction on the vector performance. All fungicides, except PE, reduced the number of YLS in nymphs treated from 3rd instar five days after starting treatments. When 3rd instar nymphs were allowed to reach adulthood, the fungicides P, PT and PC caused the highest nymphal mortality, significantly reduced the number of YLS in newly emerged adults, lengthened development time and negatively affected morphometric variables of females. There was also a trend towards a higher occurrence of brachypters. In females, the fungicides P, PT and PC caused a greater reduction of YLS when nymphs were treated to these fungicides from 3rd instar compared with nymphs treated from 4th instar. In males, the YLS number was significantly less when nymphs were fed on plants treated with P, PT, PC, and T in 3rd instar. This showed that fungicide treatments caused a greater reduction of YLS when they were applied in early stages of development. Our results provide an effective method to reduce the abundance of YLS in D. kuscheli, contributing to understand the ecological role that these symbionts could be playing in the success of this maize planthopper pest.     

NONSTATIONARY PATTERNS OF ISOLATION‐BY‐DISTANCE: INFERRING MEASURES OF LOCAL GENETIC DIFFERENTIATION WITH BAYESIAN KRIGING

Patterns of isolation‐by‐distance (IBD) arise when population differentiation increases with increasing geographic distances. Patterns of IBD are usually caused by local spatial dispersal, which explains why differences of allele frequencies between populations accumulate with distance. However, spatial variations of demographic parameters such as migration rate or population density can generate nonstationary patterns of IBD where the rate at which genetic differentiation accumulates varies across space. To characterize nonstationary patterns of IBD, we infer local genetic differentiation based on Bayesian kriging. Local genetic differentiation for a sampled population is defined as the average genetic differentiation between the sampled population and fictive neighboring populations. To avoid defining populations in advance, the method can also be applied at the scale of individuals making it relevant for landscape genetics. Inference of local genetic differentiation relies on a matrix of pairwise similarity or dissimilarity between populations or individuals such as matrices of between pairs of populations. Simulation studies show that maps of local genetic differentiation can reveal barriers to gene flow but also other patterns such as continuous variations of gene flow across habitat. The potential of the method is illustrated with two datasets: single nucleotide polymorphisms from human Swedish populations and dominant markers for alpine plant species.     

Population Dynamics and Damage Potential of Meloidogyne hapla to Rose Rootstock Species

The relationship between initial population densities (Pi) of Meloidogyne hapla on growth of three rose rootstocks (Rosa corymbifera ‘Laxa’, R. multiflora and R. canina ‘Inermis’) and nematode population development was studied. Each plant species was inoculated with ranges of nematode densities of 0, 0.062, 0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64 and 128 second‐stage juvenile/g soil and were allowed to grow for 9 weeks. Seinhorst yield model was fitted to total fresh biomass data of the rootstocks. The tolerance limits (T) were 0.04, 0.09 and 0.01 J2/g soil and the minimum yield (m) 0.65, 0.47 and 0.43 for R. corymbifera ‘Laxa’, R. multiflora and R. canina ‘Inermis’, respectively. The reproductive factor (Pf/Pi) was highest at low initial nematode densities for all rootstocks and then decreased to below maintenance level with increasing initial population densities. Root gall severity consistently increased with initial nematode population density. Furthermore, number of root galling showed a strong positive relationship with final nematode population per gram root fresh weight. The relation between Pi and Pf was also fitted to the Seinhorst population model (Pf = (M*Pi)/Pi + M/a). Rosa multiflora supported the population of M. hapla to a maximum population density (M) of 27.53 J2/g soil with an estimated average maximum multiplication rate (a) of 24.39. For R. corymbifera ‘Laxa’ and R. canina, the maximum multiplication rate was 4.34 and 3.62 and the maximum population density 6.08 and 4.78 J2/g dry soil, respectively. Hence, it was demonstrated that all three rootstocks were susceptible to even low initial nematode densities and therefore are considered good hosts for M. hapla.     

Effects of partial selfing on the equilibrium genetic variance,mutation load,and inbreeding depression under stabilizing selection

The mating system of a species is expected to have important effects on its genetic diversity. In this article, we explore the effects of partial selfing on the equilibrium genetic variance V_g, mutation load L, and inbreeding depression δ under stabilizing selection acting on a arbitrary number n of quantitative traits coded by biallelic loci with additive effects. When the ratio is low (where U is the total haploid mutation rate on selected traits) and effective recombination rates are sufficiently high, genetic associations between loci are negligible and the genetic variance, mutation load, and inbreeding depression are well predicted by approximations based on single‐locus models. For higher values of and/or lower effective recombination, moderate genetic associations generated by epistasis tend to increase V_g, L, and δ, this regime being well predicted by approximations including the effects of pairwise associations between loci. For yet higher values of and/or lower effective recombination, a different regime is reached under which the maintenance of coadapted gene complexes reduces V_g, L, and δ. Simulations indicate that the values of V_g, L, and δ are little affected by assumptions regarding the number of possible alleles per locus.     

Comparison of in vivo acute lethal potency and in vitro cytotoxicity of 48 chemicals

The cytotoxicity of 48 compounds included in the MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) list was determined in cultures of rat hepatocytes, McCoy, and MDBK cells. The average minimum concentration of each compound inducing cytotoxicity was measured in each cell type. The cytotoxicity values were then compared with published oral LDS p values for rats and mice. The logarithmic transformation of in vivo toxic doses and the corresponding in vitro cytotoxic concentrations showed a statistically significant correlation between the in vitro and in vivo values. The results show that an accurate in vivo LDS p dose could be predicted from in vitro data for at least 75% of the selected compounds. It is hoped that this finding will not only stimulate others to pursue in vitro technique but will eventually lead to elimination of the in vivo LD₅₀ test.Abbreviations CT₅₀ 50% cytotoxic concentration - CT₁₀₀ 100% cytotoxic concentration - DMSO dimethyl sulfoxide - LD₅₀ 50% lethal dose - LDH Lactate dehydrogenase - MEIC Multicenter Evaluation of In Vitro Cytotoxicity     

Effects of the IGRs compounds,lufenuron and fenoxycarb on Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae)

In this research work, the susceptibility of egg and four larval instars of Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae) to Insect Growth Regulators (IGRs) compounds (lufenuron 25% EC and fenoxycarb 25% WP) was determined. Different larval instar groups were separated by measuring the head capsule width and were used in all bioassays. The data were analysed with log-probit transformation using the SPSS software. The LC₅₀ for egg was determined by dipping egg masses in different concentration of either compound for 10 s, and LC₅₀ values for each group of larvae was estimated by using treated potato plants. The LC₅₀ values of lufenuron on egg, first, second and third instars of larvae were 682.65, 40.58, 47.83 and 261.38 ppm, respectively, and for fenoxycarb, these were estimated as 897.50, 35.60, 57.91 and 355.23 ppm, respectively. The LD₅₀ values of lufenuron and fenoxycarb on second instar larvae were 139.56 and 228.42 ppm, respectively.     

Precision of the age–length increments of three cyprinids: effects of fish number and sub‐sampling strategy

The effects of number of fish that are aged and scale sub‐sampling strategies on the precision of estimates of mean age–length increments from populations of Rutilus rutilus, Leuciscus leuciscus and Leuciscus cephalus were tested. Analyses used data derived from river fish communities in eastern England, U.K.. Regarding the number of fishes analysed in each age group, for each species and mean fork‐length increment at age, significant relationships were detected between sample size (n) and the coefficient of variation of the mean (Z) and mean length increment and measured variance (s²). This enabled calculation of the number of scales for producing a mean length increment at age according to . Outputs indicated that the number of scales requiring ageing increased substantially as precision increased, but with little variation between species per age category. Ageing between seven and 12 scales per age group would thus provide estimates at 10% precision. As the ages of fishes are not known in advance of scale ageing, the effect of scale sub‐sampling regime on precision was also tested using randomized strategies of 10 fish per 5 mm, five per 5 mm, three per 5 mm, 10 per 10 mm, five per 10 mm and three per 10 mm. These were applied to the datasets and the consequences of their reduction in the number of scales for precision were determined using . When compared to no sub‐sampling, three per 10 mm always significantly reduced data precision, whereas 10 per 5 mm never significantly reduced precision. These outputs can thus be applied to the design of fish sampling protocols where age and growth estimates are required, with the randomized sub‐sampling likely to be the most useful strategy.     

Statistical exploration of variation in quantitative two-dimensional gel electrophoresis data

Two-dimensional gel electrophoresis is a major technique in global analysis at the protein level. This paper presents an examination of spot volume data from three gel sets with radioactively labeled yeast Saccharomyces cerevisiae proteins. A strong variance versus mean dependence in data was found to be stabilized by applying a shifted logarithmic transformation. However, transformed data showed a remaining substantial variance heterogeneity for different proteins. Furthermore, examination of studentized residuals revealed that transformed data were approximately normally distributed and that there were spatial correlations among the measurement errors in the gel.     

Sampling potatoes for the incidence of tuber diseases and levels of inoculum

In three years, between three and six seed stocks of cv. Pentland Crown were planted in field experiments. Seed tubers, stem bases and progeny tubers from the growing crop and tubers at harvest and after storage were sampled intensively and estimates of inoculum and disease made for five principal tuber diseases: the blemishes skin spot (Polyscytalum pustulans), silver scurf (Helminthosporium solani) and black scurf (Rhizoctonia solani) and bacterial soft rot (Erwinia carotovora). The data were principally used to calculate sample variances after transformation of % values to logits. A variance of 0.010 was selected as giving acceptable accuracy for most purposes and to achieve such values substantial numbers of samples would have been needed. Five to ten 50-tuber samples of seed tubers or 15–20 samples of a harvested or stored crop would have been required for assessing disease symptoms and similar numbers of samples for assessing gangrene potential by a standard damage test. The eye plug test for assessing inoculum of the three blemishing diseases used 15 or 20-tuber samples and five such samples of seed or about ten from the growing crop would usually have been adequate. Variances were nearly always greater for black scurf assessments than for other blemishing diseases. The data also illustrated changes in inoculum and disease levels during the season and in store. Their significance for understanding the epidemiology of the diseases and for storage disease forecasting is discussed.     

An estimator of the Opportunity for Selection that is independent of mean fitness

Variation among individuals in number of offspring (fitness, k) sets an upper limit to the evolutionary response to selection. This constraint is quantified by Crow's Opportunity for Selection (I), which is the variance in relative fitness (I = σ²_k/(u_k)²). Crow's I has been widely used but remains controversial because it depends on mean offspring number in a sample (). Here, I used a generalized Wright-Fisher model that allows for unequal probabilities of producing offspring to evaluate behavior of Crow's I and related indices under a wide range of sampling scenarios. Analytical and numerical results are congruent and show that rescaling the sample variance (s²_k) to its expected value at a fixed removes dependence of I on mean offspring number, but the result still depends on choice of . A new index is introduced, Δ_I = Π– E(Î_drift) = Π– 1/, which makes Î independent of sample without the need for variance rescaling. Δ_I has a straightforward interpretation as the component of variance in relative fitness that exceeds that expected under a null model of random reproductive success. Δ_I can be used to directly compare estimates of the Opportunity for Selection for samples from different studies, different sexes, and different life stages.     

SMALL POPULATION GENETIC VARIABILITY AT LOCI UNDER STABILIZING SELECTION

Genetic variability at a locus under stabilizing selection in a finite population is investigated using analytic methods and computer simulations. Three measures are examined: the number of alleles k, heterozygosity H, and additive genetic variance Vg. A nearly-neutral theory results. The composite parameter S = NV_M/V_s (where N is the population size, V_M the variance of new mutant allelic effects and V_s the weakness of stabilizing selection) figures prominently in the results. The equilibrium heterozygosity is similar to that of strictly neutral theory, H = 4N_μc/ (1 + 4N_μc), except that μ_c = where c is about 0.5. Simulations corroborate except for very low N. Genetic variability attains similar equilibrium values at both a “lone” locus and at an “embedded” locus. This agrees with my earlier work concerning molecular clock rates. These results modify the neutralist interpretation of data concerning genetic variability and genetic distances between populations. Low H values are proportional not to N but to . This may explain the narrow observed range of H among species. Heterozygosities need not be highly correlated to genetic variances. Genetic variances are not highly dependent on population size except in very small populations which are difficult to sample without bias because the smallest populations go extinct the fastest. Nearly neutral evolution will not be easily distinguished from strictly neutral theory under the Hudson-Kreitman-Aguade inter-/intraspecific variation ratio test, since a similar effective mutation rate holds for genetic distances and D = 2μ_ct, where . As with strictly neutral theory, comparisons across loci should show D and H to be positively correlated because of the shared μ_c. But unlike neutral theory, for a given locus, comparisons across species should show D and H to be negatively correlated. There is no obvious threshold of population size below which genetic variability inevitably declines. Extinction depends on both genetic variation and natural selection. Neither theory nor observation presently indicates the measure of genetic variability (k, H, V_G or other) that best indicates vulnerability of a small population to extinction.     

Variance stabilization applied to microarray data calibration and to the quantification of differential expression

We introduce a statistical model for microarray gene expression data that comprises data calibration, the quantification of differential expression, and the quantification of measurement error. In particular, we derive a transformation h for intensity measurements, and a difference statistic Deltah whose variance is approximately constant along the whole intensity range. This forms a basis for statistical inference from microarray data, and provides a rational data pre-processing strategy for multivariate analyses. For the transformation h, the parametric form h(x)=arsinh(a+bx) is derived from a model of the variance-versus-mean dependence for microarray intensity data, using the method of variance stabilizing transformations. For large intensities, h coincides with the logarithmic transformation, and Deltah with the log-ratio. The parameters of h together with those of the calibration between experiments are estimated with a robust variant of maximum-likelihood estimation. We demonstrate our approach on data sets from different experimental platforms, including two-colour cDNA arrays and a series of Affymetrix oligonucleotide arrays.     

THE MAINTENANCE OF POLYGENIC VARIATION IN FINITE POPULATIONS

Models of the maintenance of genetic variance in a polygenic trait have usually assumed that population size is infinite and that selection is weak. Consequently, they will overestimate the amount of variation maintained in finite populations. I derive approximations for the equilibrium genetic variance, in finite populations under weak stabilizing selection for triallelic loci and for an infinite “rare alleles” model. These are compared to results for neutral characters, to the “Gaussian allelic” model, and to Wright's approximation for a biallelic locus under arbitrary selection pressures. For a variety of parameter values, the three-allele, Gaussian, and Wrightian approximations all converge on the neutral model when population size is small. As expected, far less equilibrium genetic variance can be maintained if effective population size, N, is on the order of a few hundred than if N is infinite. All of the models predict that comparisons among populations with N less than about 10⁴ should show substantial differences in . While it is easier to maintain absolute when alleles interact to yield dominance or overdominance for fitness, less additivity also makes more susceptible to differences in N. I argue that experimental data do not seem to reflect the predicted degree of relationship between N and . This calls into question the ability of mutation-selection balance or simple balancing selection to explain observed . The dependence of on N could be used to test the adequacy of mutation-selection balance models.     

RISK OF POPULATION EXTINCTION FROM FIXATION OF NEW DELETERIOUS MUTATIONS

The fixation of new deleterious mutations is analyzed for a randomly mating population of constant size with no environmental or demographic stochasticity. Mildly deleterious mutations are far more important in causing loss of fitness and eventual extinction than are lethal and semilethal mutations in populations with effective sizes, N_e, larger than a few individuals. If all mildly deleterious mutations have the same selection coefficient, s against heterozygotes and 2s against homozygotes, the mean time to extinction, , is asymptotically proportional to for 4N_es > 1. Nearly neutral mutations pose the greatest risk of extinction for stable populations, because the magnitude of selection coefficient that minimizes is about ? = 0.4/N_e. The influence of variance in selection coefficients among mutations is analyzed assuming a gamma distribution of s, with mean and variance . The mean time to extinction increases with variance in selection coefficients if is near ?, but can decrease greatly if is much larger than ?. For a given coefficient of variation of , the mean time to extinction is asymptotically proportional to for . When s is exponentially distributed, (c = 1) is asymptotically proportional to . These results in conjunction with data on the rate and magnitude of mildly deleterious mutations in Drosophila melanogaster indicate that even moderately large populations, with effective sizes on the order of N_e = 10³, may incur a substantial risk of extinction from the fixation of new mutations.