Stress Induces Trait Variability across Multiple Spatial Scales in the Arid Annual Plant Anastatica hierochuntica.

Variations in plant characteristics in response to habitat heterogeneity can provide valuable insights into the mechanisms governing plant responses to environmental conditions. In this study, we investigated the role of environmental factors associated with arid conditions in shaping the phenotypic responses of an arid annual plant, Anastatica hierochuntica, across several populations found along an aridity gradient and across multiple spatial scales. Utilizing both field surveys and a net house experiment, we assessed the effects of environmental factors on trait variability within and between populations. The results indicated a significant convergence in plant height due to site aridity, reflecting growth potential based on abiotic resources. Convergence was also observed in the plant's electrolyte leakage with aridity and in plant height concerning soil salinity at specific sites. Phenotypic plasticity was pivotal in maintaining trait variability, with plant height plasticity increasing with soil salinity, SLA plasticity decreasing with aridity, and leaf number plasticity rising with aridity. In conclusion, our findings underscore the adaptive significance of phenotypic variability, especially plasticity, in arid conditions. Notably, trait variability and plasticity did not consistently diminish in stressful settings, emphasizing the adaptive value of flexible responses in such environments.

An experimental design for obtaining DNA of a target species and its diet from a single non-invasive genetic protocol.

Next-generation sequencing technology has enabled accurate insights into the diet of wildlife species. The protocols for faecal sample collection and DNA extraction for diet analysis have differed from those focusing on target species, even in most studies combining questions on both aspects. We designed an experiment to evaluate two protocols using 11 parameters and select a single one that will generate both target species (Asiatic wild ass, Equus hemionus, in Israel) and diet DNA, as an effective strategy to minimise time, effort, and cost without hampering efficiency. In Protocol A, we swabbed the outer surface of faecal boluses and extracted DNA using a Stool Kit, while for Protocol B, we homogenised faecal matter from inside the bolus followed by extraction using a Powersoil Kit. Protocol A performed significantly better for four parameters, which included, for the target species, microsatellite amplification success and the quantity of the GAPDH gene; and for its diet, the number of exact sequence variants (ESVs) obtained at genus level and plant genus richness. However, there was no significant difference in the amplification success of sex-linked and plant markers, total reads at genus level, number of genera obtained and plant genus composition. Although we chose Protocol A, both protocols yielded results for the target species and its diet, demonstrating that one single protocol can be used for both purposes, although a pilot study is recommended to optimise the protocol for specific systems. This strategy may also be useful for studies combining target species and their gut microbiome and parasitic load.

Batesian mimicry or general food deception? An evolutionary game between plants for pollinator services.

Floral food deception is a well-known phenomenon which is not thoroughly understood. Particularly, it is unclear what drives a plant towards Batesian mimicry or towards generalized food deception. We analysed the evolutionary game between a Model species with nectar-secreting flowers and a Deceiver species that provides no nectar who share pollinators for reproduction. We focused our analysis on the effect of similarity of floral signals between participating plants and on costs of nectar production. We defined payoffs in the game between Models and Deceivers as the stationary visitation frequencies to participating species with different signal similarities and nectar costs. Therefore, fitness payoff of each strategy was a product of complex interactions between plant species composing the community and the pollinators visiting them. Our model provides a unified framework in which consequences of Model species interaction with different deception modes can be compared. Our findings suggest that plant-pollinator systems, like other mutualistic systems, are prone to exploitation, and that exploitation may persist at a large range of conditions. We showed that floral similarity, and thus, pollinators' ability to discriminate between Model and deceptive species, governs the stability of Batesian mimicry, while pollinator switching and sampling behaviour enables the persistence of general food deception.

Phylogeny and abiotic conditions shape the diel floral emission patterns of desert Brassicaceae species.

A key facet of floral scent is diel fluctuations in emission, often studied in the context of plant-pollinator interactions, while contributions of environment and phylogeny remain overlooked. Here, we ask if these factors are involved in shaping temporal variations in scent emission. To that end, we coupled light/dark floral emission measurements of 17 desert Brassicaceae species with environmental and phylogenetic data to explore the individual/combined impacts of these predictors on diel emission patterns. We further investigated these patterns by conducting high-resolution emission measurements in a subset of genetically distant species with contrasting temporal dynamics. While diel shifts in magnitude and richness of emission were strongly affected by genetic relatedness, they also reflect the environmental conditions under which the species grow. Specifically, light/dark emission ratios were negatively affected by an increase in winter temperatures, known to impact both plant physiology and insect locomotion, and sandy soil fractions, previously shown to exert stress that tempers with diel metabolic rhythms. Additionally, the biosynthetic origins of the compounds were associated with their corresponding production patterns, possibly to maximize emission efficacy. Using a multidisciplinary chemical/ecological approach, we uncover and differentiate the main factors shaping floral scent diel fluctuations, highlighting their consequences under changing global climate.

Ant Guild Identity Determines Seed Fate at the Post-Removal Seed Dispersal Stages of a Desert Perennial.

Ants play a dual role in their interaction with plant seeds. In deserts, the consumption of seeds by granivorous ants is common, whereas mutualistic seed dispersal, often associated with scavenging ants, is rarely documented. We evaluated the contribution of both ant guilds to efficient seed dispersal of an ant-dispersed plant, Sternbergia clusiana, in a desert ecosystem. We presented seed to colonies of three species of desert ants from the Cataglyphis (scavengers) and Messor (granivorous) genera. We recorded seed consumption, ejection from the nest, and seed transportation to potentially beneficial microhabitats. We evaluated microhabitat quality by testing the association between habitat types and the plant at various life stages. As expected, granivores mainly consumed the seeds, whereas scavengers consumed the elaiosome (seed appendage serving as a reward), but left the seeds intact. Moreover, scavenging ants relocated the seeds much further than granivores, mainly to shrub patches. The disproportional distribution of the plant under shrubs at several life stages suggests that this microhabitat is beneficial for the plant. Overall, while granivores seem to mainly harm seed dispersal, we provide the first evidence for the beneficial contribution of scavenging ants in deserts, showing they exhibit the same suite of characteristics that render them efficient seed dispersers in other ecosystems.

Reduced dispersal at nonexpanding range margins: A matter of disperser identity.

The evolution of dispersal at range margins received much attention recently, especially in the context of dynamic range shifts, such as those following climate change. However, much less attention has been devoted to study variation in and selection on dispersal at nonexpanding range margins, where populations are often small and isolated, and empirical test is dearly missing. To fill this gap, we tested whether dispersal of an ant-dispersed perennial plant (Sternbergia clusiana) is quantitatively and/or qualitatively reduced toward a nonexpanding range margin. We evaluated plant investment in dispersal structures (elaiosome), seed removal rates, and the relative abundance, activity, and behavior of low- and high-quality seed-dispersing ants in six sites ranging from mesic Mediterranean site to arid site (>600 to <100 mm of annual rainfall, respectively), which marks the southern range margin of the species. In a set of cafeteria and baiting experiments, we found that overall seed removal rates, the contribution of high-quality dispersers, maximum dispersal distance and dispersal-conducive ant behavior decreased toward range margins. These findings agree with a lower investment in reward by range margin plant populations, as reflected by lower elaiosome/seed ratio, but not by variation in the reward chemistry. More than variation in traits controlled by the plants, the variation in ant-seed interactions could be attributed to reduced presence and activity of the more efficient seed-dispersing ants in the marginal populations. Specifically, we found a mismatch between local distribution of potentially effective seed dispersers and that of the plant, even though those dispersers were observed in the study site. Interestingly, although the observed variation in the outcome of ant-seed interactions supported the prediction of reduced dispersal at nonexpanding range margins with small and isolated populations, the underlying mechanism seems to be incidental difference in the seed-dispersing ant community rather than a plant-mediated response to selection.

The combined impacts of wheat spatial position and phenology on cereal aphid abundance.

BACKGROUND: Wheat is a staple crop that suffers from massive yield losses caused by cereal aphids. Many factors can determine the abundance of cereal aphids and the damage they cause to plants; among them are the plant's genetic background, as well as environmental conditions such as spatial position within the plot, the composition and the distance from neighboring vegetation. Although the effects of these factors have been under scrutiny for many years, the combined effect of both factors on aphid populations is not fully understood. The goal of this study was to examine the collective impact of genotype and environment on wheat phenology (developmental stages), chemical diversity (metabolites), and insect susceptibility, as manifested by cereal aphid abundance. METHODS: To determine the influence of plant genotype on the metrics mentioned above, we measured the phenology, chemical profile, and aphid abundance of four wheat genotypes, including the tetraploid wild emmer (Triticum turgidum ssp. dicoccoides cv. Zavitan), tetraploid durum (Triticum turgidum ssp. durum cv. Svevo), and two hexaploid spring bread (Triticum aestivum), 'Rotem' and 'Chinese Spring'. These genotypes are referred to as "focal" plants. To evaluate the impact of the environment, we scored the distance of each focal plant (spatial position) from two neighboring vegetation types: (i) natural resource and (ii) monoculture wheat resource. RESULTS: The results demonstrated that the wild emmer wheat was the most aphid-resistant, while the bread wheat Rotem was most aphid-susceptible. Aphids were more abundant in plants that matured early. The spatial position analysis demonstrated that aphids were more abundant in focal plants located closer to the margin monoculture wheat resource rather than to the natural resource, suggesting a resource concentration effect. The analysis of metabolic diversity showed that the levels of three specialized metabolites from the flavonoid class, differed between the wheat genotypes and some minor changes in central metabolites were shown as well. Altogether, these results demonstrate a combined effect of genetic background and spatial position on wheat phenology and aphid abundance on plants. This exposes the potential role of the marginal vegetation environment in shaping the insect population of desirable crops. These findings highlight the importance of maintaining plant intra-specific variation in the agriculture system because of its potential applications in reducing pest density.

Period doubling as an indicator for ecosystem sensitivity to climate extremes.

The predictions for a warmer and drier climate and for increased likelihood of climate extremes raise high concerns about the possible collapse of dryland ecosystems, and about the formation of new drylands where native species are less tolerant to water stress. Using a dryland-vegetation model for plant species that display different tradeoffs between fast growth and tolerance to droughts, we find that ecosystems subjected to strong seasonal variability, typical for drylands, exhibit a temporal period-doubling route to chaos that results in early collapse to bare soil. We further find that fast-growing plants go through period doubling sooner and span wider chaotic ranges than stress-tolerant plants. We propose the detection of period-doubling signatures in power spectra as early indicators of ecosystem collapse that outperform existing indicators in their ability to warn against climate extremes and capture the heightened vulnerability of newly-formed drylands. The proposed indicator is expected to apply to other types of ecosystems, such as consumer-resource and predator-prey systems. We conclude by delineating the conditions ecosystems should meet in order for the proposed indicator to apply.

Investment in reward by ant-dispersed plants consistently selects for better partners along a geographic gradient.

Seed dispersal by ants (myrmecochory) is an asymmetric, presumably mutualistic interaction, where a few ant species benefit many plants. Myrmecochorous plants express specialized adaptations, most notably a large elaiosome, which promote interactions with efficient seed dispersers while decreasing interactions with poor dispersers, resulting in de facto partner choice. However, because variation in plants' investment in reward and ant response to them may vary spatially and temporally, it is unclear whether such specialization is consistent along geographic gradients; especially towards myrmecochory's range margin. To answer this question on context-dependent partner choice, we first estimated variation in reward investment by co-occurring myrmecochores along a steep environmental gradient in a Mediterranean region. Second, we tested whether variation in plant investment in reward was positively and consistently correlated with the quality of dispersal plant received along the same gradient. Using in situ cafeteria experiments, we simultaneously presented diaspores of locally co-occurring myrmecochorous species to ants of two guilds representing high- and low-quality dispersers. We then recorded ant-seed behaviour, seed preference and seed removal rates for each ant guild. We found both overall and within-site high variation among plant species in the total and relative investment in elaiosomes. Both ant guilds removed substantial proportion of the seeds. However, scavenging ants (high-quality dispersers) clearly preferred diaspores with larger elaiosomes, whereas granivorous ants (low-quality dispersers) exhibited no preference. Furthermore, both the variation in plant traits and the corresponding response of different ant guilds were consistent along the studied geographic gradient. This consistency holds even when granivores, which removed seeds in a non-selective fashion and provided apparently low-quality seed dispersal services, were, at least numerically, the dominant ant guild. This dominance and the consistency of the partner choice shed light on the functionality of elaiosomes at the margins of myrmecochory's distribution.

Fine-scale substrate heterogeneity does not affect arthropod communities on green roofs.

Green roofs, which are roofs with growing substrate and vegetation, can provide habitat for arthropods in cities. Maintaining a diversity of arthropods in an urban environment can enhance the functions they fill, such as pest control and soil development. Theory suggests that the creation of a heterogeneous environment on green roofs would enhance arthropod diversity. Several studies have examined how arthropod diversity can be enhanced on green roofs, and particularly whether substrate properties affect the arthropod community, but a gap remains in identifying the effect of substrate heterogeneity within a green roof on the arthropod community. In this paper, it is hypothesized that creating heterogeneity in the substrate would directly affect the diversity and abundance of some arthropod taxa, and indirectly increase arthropod diversity through increased plant diversity. These hypotheses were tested using green roof plots in four treatments of substrate heterogeneity: (1) homogeneous dispersion; (2) mineral heterogeneity-with increased tuff concentration in subplots; (3) organic heterogeneity-with decreased compost concentrations in subplots; (4) both mineral and organic heterogeneity. Each of the four treatments was replicated twice on each of three roofs (six replicates per treatment) in a Mediterranean region. There was no effect of substrate heterogeneity on arthropod diversity, abundance, or community composition, but there were differences in arthropod communities among roofs. This suggests that the location of a green roof, which can differ in local climatic conditions, can have a strong effect on the composition of the arthropod community. Thus, arthropod diversity may be promoted by building green roofs in a variety of locations throughout a city, even if the roof construction is similar on all roofs.

Interplay between exogenous and endogenous factors in seasonal vegetation oscillations.

A fundamental question in ecology is whether vegetation oscillations are merely a result of periodic environmental variability, or rather driven by endogenous factors. We address this question using a mathematical model of dryland vegetation subjected to annual rainfall periodicity. We show that while spontaneous oscillations do not exist in realistic parameter ranges, resonant response to periodic precipitation is still possible due to the existence of damped oscillatory modes. Using multiple time-scale analysis, in a restricted parameter range, we find that these endogenous modes can be pumped by the exogenous precipitation forcing to form sustained oscillations. The oscillations amplitude shows a resonance peak that depends on model parameters representing species traits and mean annual precipitation. Extending the study to bistability ranges of uniform vegetation and bare soil, we investigate numerically the implications of resonant oscillations for ecosystem function. We consider trait parameters that represent species with damped oscillatory modes and species that lack such modes, and compare their behaviors. We find that the former are less resilient to droughts, suffer from larger declines in their biomass production as the precipitation amplitude is increased, and, in the presence of spatial disturbances, are likely to go through abrupt collapse to bare soil, rather than gradual, domino-like collapse.

Green roof and photovoltaic panel integration: Effects on plant and arthropod diversity and electricity production.

The combination of green roofs with photovoltaic (PV) panels has been proposed to provide synergistic benefits as the panel is cooled by the presence of the vegetation, and thus produces more electricity, while the solar panel enhances growing conditions for vegetation, and increases abiotic heterogeneity, resulting in higher plant diversity. We tested these hypotheses in a non-irrigated green roof in a Mediterranean climate with replicated plots including green roofs only, green roofs with a PV panel, and a conventional roof surface with a PV panel. We found that presence of a panel resulted in higher heterogeneity in substrate moisture, but there was no effect on plant diversity. Plant species showed enhanced growth in plots with PV, including greater growth of Sedum sediforme and longer flowering time of annual species. On the other hand, arthropod diversity was lower during part of the year, and abundance of some arthropod taxa was lower in green roof plots with PV. The presence of the green roof also did not improve electricity production by the panels. We conclude that in a Mediterranean climate, it would be appropriate to examine the use of irrigation in green roofs with PV panels, including effects on the plant community and on electricity production.

Insights into root structure and function of Bassia indica: water redistribution and element dispersion.

In the last few decades, research has increasingly been aimed at clarifying how root system architecture, physiology and function are related to environmental drivers. 'Negative halotropism' has been defined as the alteration of root growth direction to avoid salinity. We suggested that 'positive halotropism' may be found in halophytes relying on salinity for optimal growth. Investigating root structure of the halophyte Bassia indica (Wight) A. J. Scott, we have shown that positive halotropism can explain the growth of horizontal roots towards optimal salt concentrations along a soil salinity gradient. Here we tested three hypotheses. First, that development of B. indica roots depends on a trade-off between optimal nutrient supply and saline concentrations: results of split-root-experiment showed a preference for sand enriched with nutrients and poor in salts. Second, that shallow horizontal roots enable B. indica to forage for nutrient-rich patches. Results demonstrated that bulk elemental analysis was not consistent with tissue-specific elemental analysis, and this can be explained by substantial variability of element composition of particular root segments. Third, we hypothesised that B. indica redistributes water horizontally through shallow horizontal roots. Results showed that back flow of water from the tap root towards tip root was possible in horizontal roots in saline microenvironment.

Symmetry and range limits in importance indices.

Recently, Mingo has analyzed the properties of I imp, an importance index, and demonstrated that its range is not symmetrical. While agreeing with this comment, we believe that more light needs to be shed on the issue of symmetry in relation to such indices. Importance indices are calculated using three values: performance of the organism in the absence and in the presence of neighbors and maximum performance of the organism in ideal conditions. Because of this structure, importance indices can hardly ever achieve symmetry along the whole range of potential performances. We discuss the limitation of the symmetry range for different symmetry types and for both additive and multiplicative indices. We conclude that importance indices, as other interactions indices, are practical tools for interpreting ecological outcomes, especially while comparing between studies. Nevertheless, the current structure of importance indices prevents symmetry along their whole range. While the lack of "perfect" symmetry may call for the development of more sophisticated importance metrics, the current indices are still helpful for the understanding of biological systems and should not be discarded before better alternatives are well established. To prevent potential confusion, we suggest that ecologists present the relevant index symmetry range in addition to their results, thus minimizing the probability of misinterpretation.

A comprehensive test of evolutionarily increased competitive ability in a highly invasive plant species.

BACKGROUND AND AIMS: A common hypothesis to explain plants' invasive success is that release from natural enemies in the introduced range selects for reduced allocation to resistance traits and a subsequent increase in resources available for growth and competitive ability (evolution of increased competitive ability, EICA). However, studies that have investigated this hypothesis have been incomplete as they either did not test for all aspects of competitive ability or did not select appropriate competitors. METHODS: Here, the prediction of increased competitive ability was examined with the invasive plant Lythrum salicaria (purple loosestrife) in a set of common-garden experiments that addressed these aspects by carefully distinguishing between competitive effect and response of invasive and native plants, and by using both intraspecific and interspecific competition settings with a highly vigorous neighbour, Urtica dioica (stinging nettle), which occurs in both ranges. KEY RESULTS: While the intraspecific competition results showed no differences in competitive effect or response between native and invasive plants, the interspecific competition experiment revealed greater competitive response and effect of invasive plants in both biomass and seed production. CONCLUSIONS: The use of both intra- and interspecific competition experiments in this study revealed opposing results. While the first experiment refutes the EICA hypothesis, the second shows strong support for it, suggesting evolutionarily increased competitive ability in invasive populations of L. salicaria. It is suggested that the use of naturally co-occurring heterospecifics, rather than conspecifics, may provide a better evaluation of the possible evolutionary shift towards greater competitive ability.

Beyond the competition-colonization trade-off: linking multiple trait response to disturbance characteristics.

Disturbances' role in shaping communities is well documented but highly disputed. We suggest replacing the overused two-trait trade-off approach with a functional group scheme, constructed from combinations of four key traits that represent four classes of species' responses to disturbances. Using model results and field observations from sites affected by two highly different disturbances, we demonstrated that popular dichotomous trade-offs are not sufficient to explain community dynamics, even if some emerge under certain conditions. Without disturbances, competition was only sufficient to predict species survival but not relative success, which required some escape mechanism (e.g., long-term dormancy). With highly predictable and large-scale disturbances, successful species showed a combination of high individual tolerance to disturbance and, more surprisingly, high competitive ability. When disturbances were less predictable, high individual tolerance and long-term seed dormancy were favored, due to higher environmental uncertainty. Our study demonstrates that theories relying on a small number of predefined trade-offs among traits (e.g., competition-colonization trade-off) may lead to unrealistic results. We suggest that the understanding of disturbance-community relationships can be significantly improved by employing sets of relevant trait assemblies instead of the currently common approach in which trade-offs are assumed in advance.

Solar radiation stress in climbing snails: behavioural and intrinsic features define the Hsp70 level in natural populations of Xeropicta derbentina (Pulmonata).

Ectotherms from sunny and hot environments need to cope with solar radiation. Mediterranean land snails of the superfamily Helicoidea feature a behavioural strategy to escape from solar radiation-induced excessive soil heating by climbing up vertical objects. The height of climbing, and also other parameters like shell colouration pattern, shell orientation, shell size, body mass, actual internal and shell surface temperature, and the interactions between those factors may be expected to modulate proteotoxic effects in snails exposed to solar radiation and, thus, their stress response. Focussing on natural populations of Xeropicta derbentina, we conducted a 'snapshot' field study using the individual Hsp70 level as a proxy for proteotoxic stress. In addition to correlation analyses, an IT-model selection approach based on Akaike's Information Criterion was applied to evaluate a set of models with respect to their explanatory power and to assess the relevance of each of the above-mentioned parameters for individual stress, by model averaging and parameter estimation. The analysis revealed particular importance of the individuals' shell size, height above ground, the shell colouration pattern and the interaction height × orientation. Our study showed that a distinct set of behavioural traits and intrinsic characters define the Hsp70 level and that environmental factors and individual features strongly interact.

Effects of age and size in the ears of gekkotan lizards: auditory sensitivity, its determinants, and new insights into tetrapod middle-ear function.

Do related, differently sized species differ in size-related structural or functional traits merely because they mature at different points of a uniform allometric ontogenetic growth curve, or do they evolutionarily diverge? We tested ears of gekkotan lizards through experiments distinguishing the two. Auditory sensitivity was assessed by compound action potential (CAP) thresholds in eight species. The best thresholds characterizing species ranged 22-72 dB sound pressure level at 0.5-1.0 kHz. Direct acoustic stimulation of the columella footplate elevated thresholds by 25-50 dB. Intraspecific CAP sensitivity was primarily affected by body length in Eublepharis macularius, but by tympanic-membrane velocity in Oedura marmorata. The chief factor determining middle-ear function (difference in CAP sensitivity before and after middle-ear ablation) was body length in both species. A secondary factor was the middle-ear hydraulic lever ratio in E. macularius, but the mechanical lever in O. marmorata. When intra- and interspecific data were compared, the relation of CAP thresholds to body size in E. macularius resembled the interspecific regression. The intraspecific regression of auditory sensitivity over tympanic membrane velocity in O. marmorata differed from that calculated interspecifically. Hence, the evolutionary contribution to size effects on CAP sensitivity exceeds the ontogenetic contribution. Putatively, body length affects CAP sensitivity through absolute sizes of tympanic membrane and columella footplate. These newly discovered effects join those of the hydraulic lever and (interspecifically) hair-cell number to improve the hearing of larger species that vocally communicate across wider spaces, apparently throughout the Tetrapoda.

Effects of age and size in the ears of gekkonomorph lizards: middle-ear morphology with evolutionary implications.

The function of the ear depends in part on its absolute size and internal proportions. Thus, in both young individuals and small species, the middle ear is expected to be allometrically enlarged despite its smaller absolute size. Here we aim to compare the ontogenetic allometry of relevant middle-ear structures as observed within gecko (gekkonomorph lizards) species, with the evolutionary allometry observed interspecifically. These observations also provide middle-ear data for future evaluation of variation in auditory sensitivity. The material comprised 84 museum specimens of geckos, representing nine species of three gekkonomorph subfamilies. The results of dissections and measurements show that different reports notwithstanding, the middle-ear ossicular chain is indeed structured as described for geckos by Werner and Wever. Some sexual dimorphism is indicated, but this requires further study. During postnatal ontogeny, the allometric growth in the ratio of the columellar footplate area to body length differed between the intraspecific and interspecific levels, hence species differences in the middle ear do not merely result from animal size. The ratio of the tympanic membrane area to the columellar footplate area increased during ontogeny. In this, geckos resemble birds and probably also mammals. Similarly, when the comparison was among adults representing different species, the ratio of the tympanic membrane area to the columellar footplate area increased with body size. In this, however, the geckos differed from birds and mammals, in which this ratio varied taxonomically, irrespective of body size. It would thus seem that middle-ear proportions have evolved among geckos to produce small interspecific differences, but among amniote tetrapods they have evolved according to different principles in the classes reptiles, birds, and mammals.