Nectar secondary compounds affect self-pollen transfer: implications for female and male reproduction

Pollen movement within and among plants affects inbreeding, plant fitness, and the spatial scale of genetic differentiation. Although a number of studies have assessed how plant and floral traits influence pollen movement via changes in pollinator behavior, few have explored how nectar chemical composition affects pollen transfer. As many as 55% of plants produce secondary compounds in their nectar, which is surprising given that nectar is typically thought to attract pollinators. We tested the hypothesis that nectar with secondary compounds may benefit plants by encouraging pollinators to leave plants after visiting only a few flowers, thus reducing self-pollen transfer. We used Gelsemium sempervirens, a plant whose nectar contains the alkaloid gelsemine, which has been shown to be a deterrent to foraging bee pollinators. We found that high nectar alkaloids reduced the total and proportion of self-pollen received by one-half and one-third, respectively. However, nectar alkaloids did not affect female reproduction when we removed the potential for self-pollination (by emasculating all flowers on plants). We then tested the assumption that self-pollen in combination with outcrossed pollen depresses seed set. We found that plants were weakly self-compatible, but self-pollen with outcrossed pollen did not reduce seed set relative to solely outcrossed flowers. Finally, an exponential model of pollen carryover suggests that high nectar alkaloids could benefit plants via increased pollen export (an estimate of male function), but only when pollinators were efficient and abundant and plants had large floral displays. Results suggest that high nectar alkaloids may benefit plants via increased pollen export under a restricted set of ecological conditions, but in general, the costs of high nectar alkaloids in reducing pollination balanced or outweighed the benefits of reducing self-pollen transfer for estimates of female and male reproduction.     

Fine-scale flower-visiting behavior revealed by using a high-speed camera

Most estimations of the pollination efficiency of insects have been based on observation by the naked human eye. However, insect behaviors are often too rapid to analyze sufficiently this way. Here we demonstrate the use of high-speed cameras to analyze the fine-scale behaviors of Macroglossum pyrrhosticta, Xylocopa appendiculata, and Papilio dehaanii when visiting Clerodendrum trichotomum. The fine-scale nectar drinking time, number of contacts with anthers and/or stigmas, and frequencies of body part contact with anthers and/or stigmas differed significantly among pollinator species. Pollination efficiency was not equal among pollinators. In addition, M. pyrrhosticta made the least number of contacts with anthers and/or stigmas even though it showed the highest visitation frequency. These results demonstrate that when examined from the viewpoint of rapid visitation behaviors, pollination dynamics differ among pollinator species, and flower visits and pollination rates are not equal.     

Pollination in Dianthus deltoides (Caryophyllaceae): Effects of Habitat Fragmentation on Visitation and Seed Set

Abstract: I analyse the effects of habitat fragmentation on the pollination success of a perennial, butterfly-pollinated, caryophyllaceous herb, the maiden pink, Dianthus deltoides L. The study was conducted in July 1986 and July 1987 at two different sites in southwest Sweden, an undisturbed "mainland" site and a fragmented site consisting of "habitat islands" within a heavily utilized agricultural area The fragmented area had a lower diversity and abundance of both flowering plants and flower-visiting insects. Dianthus flowers received fewer visits in the fragmented area than in the mainland area, and the seed set was much lower. Hand pollination increased seed set up to 4.1 times in the fragmented area, but no significant differences were found between hand-pollinated and control flowers at the mainland site. There were no differences between the two sites in standing crop of nectar, ovule number per flowers, or seed set of bagged flowers, band-pollinated flowers, and hand-pollinated fertilized flowers Thus, the difference in natural seed set between the two sites can be explained by differences in pollinator service.     

Imperfect Replacement of Native Species by Non‐Native Species as Pollinators of Endemic Hawaiian Plants

Native plant species that have lost their mutualist partners may require non‐native pollinators or seed dispersers to maintain reproduction. When natives are highly specialized, however, it appears doubtful that introduced generalists will partner effectively with them. We used visitation observations and pollination treatments (experimental manipulations of pollen transfer) to examine relationships between the introduced, generalist Japanese White‐eye (Zosterops japonicus) and 3 endemic Hawaiian plant species (Clermontia parviflora, C. montis‐loa, and C. hawaiiensis). These plants are characterized by curved, tubular flowers, apparently adapted for pollination by curve‐billed Hawaiian honeycreepers. Z. japonicus were responsible for over 80% of visits to flowers of the small‐flowered C. parviflora and the midsize‐flowered C. montis‐loa. Z. japonicus‐visited flowers set significantly more seed than did bagged flowers. Z. japonicus also demonstrated the potential to act as an occasional Clermontia seed disperser, although ground‐based frugivory by non‐native mammals likely dominates seed dispersal. The large‐flowered C. hawaiiensis received no visitation by any birds during observations. Unmanipulated and bagged C. hawaiiensis flowers set similar numbers of seeds. Direct examination of Z. japonicus and Clermontia morphologies suggests a mismatch between Z. japonicus bill morphology and C. hawaiiensis flower morphology. In combination, our results suggest that Z. japonicus has established an effective pollination relationship with C. parviflora and C. montis‐loa and that the large flowers of C. hawaiiensis preclude effective visitation by Z. japonicus. Remplazo Imperfecto de Especies Nativas por Especies No‐Nativas como Polinizadores de Plantas Endémicas de Hawaii     

Higher soybean production using honeybee and wild pollinators, a sustainable alternative to pesticides and autopollination

This is the first report showing that using honeybee (Apis mellifera) and wild pollinators complementary pollination can enhance soybean productivity (Glycine max). Current industrial production of soybean involves autopollination and high loads of pesticides. Therefore, growers have neglected possible biotic pollination despite suggestions that soybean benefit from insect pollinators. Reports advocating possible biotic pollination are based on experiments where bees are caged with flowering plants and the absence of pesticides, thus not in field conditions. Therefore, here we compared in field conditions soybean yield produced (1) independently of biotic pollinators, (2) with wild pollinators and (3) with honeybee colonies. Results showed an increase of +6.34 % of soybean yield in areas where wild pollinators had free access to flowers. The introduction of honeybee colonies further raised the yield of +18.09 %. Our findings therefore show that, though soybean is autogamous, allowing pollination by wild pollinators leads to higher yields. Moreover, adding honeybee mitigates pollination deficits and improves yield compared to current practices.     

Mimics and magnets: the importance of color and ecological facilitation in floral deception

Plants that lack floral rewards can attract pollinators if they share attractive floral signals with rewarding plants. These deceptive plants should benefit from flowering in close proximity to such rewarding plants, because pollinators are locally conditioned on floral signals of the rewarding plants (mimic effect) and because pollinators are more abundant close to rewarding plants (magnet effect). We tested these ideas using the non-rewarding South African plant Eulophia zeyheriana (Orchidaceae) as a study system. Field observations revealed that E. zeyheriana is pollinated solely by solitary bees belonging to a single species of Lipotriches (Halictidae) that appears to be closely associated with the flowers of Wahlenbergia cuspidata (Campanulaceae), a rewarding plant with which the orchid is often sympatric. The pale blue color of the flowers of E. zeyheriana differs strongly from flowers of its congeners, but is very similar to that of flowers of W. cuspidata. Analysis of spectral reflectance patterns using a bee vision model showed that bees are unlikely to be able to distinguish the two species in terms of flower color. A UV-absorbing sunscreen was applied to the flowers of the orchid in order to alter their color, and this resulted in a significant decline in pollinator visits, thus indicating the importance of flower color for attraction of Lipotriches bees. Pollination success in the orchid was strongly affected by proximity to patches of W. cuspidata. This was evident from one of two surveys of natural populations of the orchid, as well as experiments in which we translocated inflorescences of the orchid either into patches of W. cuspidata or 40 m outside such patches. Flower color and location of E. zeyheriana plants relative to rewarding magnet patches are therefore key components of the exploitation by this orchid of the relationship between W. cuspidata and Lipotriches bee pollinators.     

Is flower scent influencing host plant selection of leaf-galling sawflies (Hymenoptera, Tenthredinidae) on willows?

Though it is known that flower scent not only attracts pollinators but also herbivores, little is known about the importance of flower scent on the distribution of leaf herbivores among individuals within a plant species. In this study we determined the distribution of galls induced by the sawfly Pontania proxima (Serville 1823) (Hymenoptera, Tenthredinidae, Nematinae) on flowering and non-flowering representatives of several clones belonging to Salix fragilis and S. × rubens (Salicaceae). Further, amounts and composition of scent of flowering and non-flowering twigs were compared (dynamic headspace-gas chromatography–mass spectrometry, DHS-GC–MS), and a scent sample collected from flowering twigs of S. fragilis was tested by coupled gas chromatography and electroantennographic detection (GC-EAD) on the antennae of P. proxima females. The results show that the presence of flower catkins on plants led to a higher degree of allocation with galls, but the number of galls differed not between flowering and non-flowering plants. The DHS-GC–MS analyses revealed that the total amount of flower scent emitted per flowering twig is about 90 times higher than the scent emitted by a non-flowering twig. Further, several compounds were emitted only by flowering but not by non-flowering twigs. In the GC-EAD analyses, antennae consistently responded not only to green leaf volatiles, but also to compounds emitted only by the flowers (e.g. 1,4-dimethoxybenzene). These flower scent compounds are suggested to affect the host plant choice by attracting more sawflies from the distance to flowering plants compared to non-flowering plants. The EAD-active compounds emitted from vegetative plant parts are assumed to act as long-distance signals especially when flowers are absent on host plants, e.g. during the oviposition period of the second generation of P. proxima.     

Net energetic advantage drives honey bees (Apis mellifera L) to nectar larceny in Vaccinium ashei Reade

Carpenter bees (Xylocopa spp.) act as primary nectar thieves in rabbiteye blueberry (Vaccinium ashei Reade), piercing corollas laterally to imbibe nectar at basal nectaries. Honey bees (Apis mellifera L) learn to visit these perforations and thus become secondary nectar thieves. We tested the hypothesis that honey bees make this behavioral switch in response to an energetic advantage realized by nectar-robbing flower visits. Nectar volume and sugar quantity were higher in intact than perforated flowers, but bees (robbers) visiting perforated flowers were able to extract a higher percentage of available nectar and sugar so that absolute amount of sugar (mg) removed by one bee visit is the same for each flower type. However, because perforated flowers facilitate higher rates of bee flower visitation and the same or higher rates of nectar ingestion, they are rendered more profitable than intact flowers in temporal terms. Accordingly, net energy (J) gain per second flower handling time was higher for robbers on most days sampled. We conclude that the majority evidence indicates an energetic advantage for honey bees that engage in secondary nectar thievery in V. ashei.Communicated by R. Page     

Anther-mimicking floral guides exploit a conflict between innate preference and learning in bumblebees (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)

Bee-pollinated plants are frequently dichogamous: e.g. each flower has a discernable male and female phase, with only the male phase offering a pollen reward. Pollen-collecting bees should therefore discriminate against female-phase flowers to maximise their rate of pollen harvest, but this behaviour would reduce plant fitness due to inferior pollination. Here, we test the hypothesis that flowers use pollen-mimicking floral guides to prevent flower-phase discrimination. Such floral guides resemble pollen in spectral reflection properties and are widespread among angiosperm flowers. In an array of artificial flowers, bumblebees learned less well to discriminate between flower variants simulating different flowering phases when both flower variants carried an additional pollen-yellow guide mark. This effect depended crucially on the pollen-yellow colour of the guide mark and on its spatial position within the artificial flower. We suggest that floral guides evolved to inhibit flower-phase learning in bees by exploiting the innate colour preferences of their pollinators.     

Effects of Pollinator Loss on Endemic New Zealand Mistletoes (Loranthaceae)

Abstract: The endemic mistletoes Peraxilla colensoi and P. tetrapetala (Loranthaceae) have declined considerably in New Zealand since 1840, reputedly because of introduced herbivores but coincident with a major decline in native bird densities. We show that at two South Island sites ( Craigieburn and Ohau) there are too few bird pollinators visiting the flowers to allow full fruit set. We studied pollination rates in P. colensoi at Wakefield and P. tetrapetala at Craigieburn over four flowering seasons and P. tetrapetala in one season at Ohau. Supplemental hand pollination increased fruit production 1.25–5.3 times at Craigieburn and Ohau but not at Wakefield. Excluding birds by covering mistletoes with mesh bags decreased fruit set significantly at Wakefield but had little effect at Craigieburn and Ohau. Bellbirds ( Anthornis melanura ) and Tuis (   Prosthemadera novaeseelandiae ) visited flowers significantly more often at Wakefield than at Craigieburn. A lack of pollen tubes in the style, not self-incompatibility or resource shortage, caused the low fruit production in unmanipulated flowers at Craigieburn. Thus, at the two P. tetrapetala sites (Craigieburn and Ohau) fruit set was chronically pollen limited, whereas P. colensoi at Wakefield was not pollen-limited. Data from other Peraxilla sites also suggest pollination failure. Our study suggests that the conservation of Peraxilla species will require maintenance of native bird populations. Tuis and Bellbirds are important pollinators and dispersers of many other New Zealand plants, and the breakdown of such mutualistic relationships may have widespread consequences.     

Pollinator effectiveness varies with experimental shifts in flowering time

The earlier flowering times exhibited by many plant species are a conspicuous sign of climate change. Altered phenologies have caused concern that species could suffer population declines if they flower at times when effective pollinators are unavailable. For two perennial wildflowers, Tradescantia ohiensis and Asclepias incarnata, we used an experimental approach to explore how changing phenology affects the taxonomic composition of the pollinator assemblage and the effectiveness of individual pollinator taxa. After finding in the previous year that fruit set varied with flowering time, we manipulated flowering onset in greenhouses, placed plants in the field over the span of five weeks, and measured pollinator effectiveness as the number of seeds produced after a single visit to a flower. The average effectiveness of pollinators and the expected rates of pollination success were lower for plants of both species flowering earlier than for plants flowering at historical times, suggesting there could be reproductive costs to earlier flowering. Whereas for A. incarnata, differences in average seed set among weeks were due primarily to changes in the composition of the pollinator assemblage, the differences for T. ohiensis were driven by the combined effects of compositional changes and increases over time in the effectiveness of some pollinator taxa. Both species face the possibility of temporal mismatch between the availability of the most effective pollinators and the onset of flowering, and changes in the effectiveness of individual pollinator taxa through time may add an unexpected element to the reproductive consequences of such mismatches.     

Indirect Effects of Pandemic Deer Overabundance Inferred from Caterpillar‐Host Relations

Externally feeding phytophagous insect larvae (i.e., caterpillars, here, larval Lepidoptera and sawflies, Hymenoptera: Symphyta) are important canopy herbivores and prey resources in temperate deciduous forests. However, composition of forest trees has changed dramatically in the eastern United States since 1900. In particular, browsing by high densities of white‐tailed deer (Odocoileus virginianus) has resulted in forests dominated by browse‐tolerant species, such as black cherry (Prunus serotina), and greatly reduced relative abundance of other tree species, notably pin cherry (Prunus pensylvanica) and birches (Betula spp.). To quantify effects of these changes on caterpillars, we sampled caterpillars from 960 branch tips of the 8 tree species that comprise 95% of trees in Allegheny hardwood forests: red maple (Acer rubrum), striped maple (Acer pensylvanicum), sugar maple (Acer saccharum), sweet birch (Betula lenta), yellow birch (Betula allegheniensis), American beech (Fagus grandifolia), black cherry, and pin cherry. We collected 547 caterpillar specimens that belonged to 66 Lepidoptera and 10 Hymenoptera species. Caterpillar density, species richness, and community composition differed significantly among tree species sampled. Pin cherry, nearly eliminated at high deer density, had the highest density and diversity of caterpillars. Pin cherry shared a common caterpillar community with black cherry, which was distinct from those of other tree hosts. As high deer density continues to replace diverse forests of cherries, maples, birches, and beech with monodominant stands of black cherry, up to 66% of caterpillar species may be eliminated. Hence, deer‐induced changes in forest vegetation are likely to ricochet back up forest food webs and therefore negatively affect species that depend on caterpillars and moths for food and pollination. Efectos Indirectos de la Sobreabundancia de Venados Pandémicos Inferida de Relaciones Orugas‐Huéspedes     

Risk aversion in hand-reared bananaquits

Summary To study risk aversion in hand-reared bananaquits (Coereba flaveola) we placed individuals in a cage with a 1 m² floral board having a random array of 85 yellow and 85 red artificial flowers. Flowers of one color were filled with the same quantity of nectar (constant flowers), whereas flowers of the other color were filled with variable quantities of nectar (variable flowers). The constant and variable flowers had identical mean contents, only their variances differed. After three presentations, the constant flowers were made variable and vice versa to control for color preferences. Naive foragers tended to avoid variable flowers. The degree of risk aversion was influenced by previous experience, the relative variability of the variable flowers, and flower color. Variable flowers having similar coefficients of variation, but different reward variables (volume or concentration) resulted in similar levels of risk aversion. Within single foraging episodes the following was observed: sequences of constant flowers increased while sequences of variable flowers remained similar to random foraging; the probability of revisiting a constant flower was higher than revisiting a variable flower; the average amount of nectar consumed from constant and variable flowers was similar within the assessment periods (prior to favoring constant flowers); the proportion of visits falling below the mean expected reward during the assessment period or its inverse (the proportion visited with at least the equivalent of the mean) may be a cue used for risk aversion; risk aversion persisted through long foraging bouts despite changed nectar distributions suggesting that the bananaquits did not track resource distributions well within foraging bouts.     

Collapse of a pollination web in small conservation areas

A suspected global decline in pollinators has heightened interest in their ecological significance. In a worst-case scenario, the decline of generalist pollinators is predicted to trigger cascades of linked declines among the multiple specialist plant species to which they are linked, but this has not been documented. I studied a portion of a pollination web involving a generalist pollinator, the oil-collecting bee Rediviva peringueyi, and a community of oil-secreting plants. Across 27 established conservation areas located in the Cape Floral Region, I found substantial variation in the bees' occurrence in relation to soil type and the successional stage of the vegetation. Anthropogenic declines were detectable against this background of naturally occurring variation: R. peringueyi was absent from small conservation areas (< 385 ha) in an urban matrix. In the absence of the bee, seed set failed in six specialist plant species that are pollinated only by R. peringueyi but remained high in a pollination generalist, which had replacement pollinators. The findings are consistent with theoretical predictions of the importance of generalist pollinators in maintaining the structure of pollination webs.     

Effects of energy requirements and worker mortality on colony growth and foraging in the honey bee

Summary A model of colony growth and foraging in the honey bee (Apis mellifera L.) is presented. It is assumed that summer workers choose a foraging strategy that maximizes colony population by the end of the season subject to the constraint that enough nectar has been stored to sustain the adult population overwinter. The optimal foraging strategy is derived with respect to the number of flowers visited during one foraging trip. A forager that visits many flowers collects a substantial amount of nectar but the probability that the worker returns alive from the excursion decreases accordingly. Using dynamic modelling, I explore the effects on colony growth of colony population, colony energy requirements and mortality rate while foraging. The model shows that when the expected rate of increase in nectar reserves is low, for instance in small colonies or when mortality rate rises rapidly with foraging intensity, workers collect more nectar during each foraging trip. The increase in foraging activity is realized at the expense of colony growth. The main finding is that depending on colony status the foraging strategy that maximizes worker population implies visits to almost any number of flowers. This is in sharp contrast to predictions from traditional foraging models where foraging intensity is assumed to cluster around values that maximize net rate or efficiency. The model suggests that strategies that cluster around rate and efficiency maximization should be viewed as particular solutions to a more general problem.     

Habitat fragmentation and effects of herbivore (howler monkey) abundances on bird species richness

Habitat fragmentation can alter herbivore abundances, potentially causing changes in the plant community that can propagate through the food web and eventually influence other important taxonomic groups such as birds. Here we test the relationship between the density of red howler monkeys (Alouatta seniculus) and bird species richness on a large set of recently isolated land-bridge islands in Lago Guri, Venezuela (n = 29 islands). Several of these islands host relict populations of howler monkeys at densities up to more than 30 times greater than those on the mainland. These "hyperabundant" herbivores previously have been shown to have a strong positive influence on aboveground plant productivity. We predicted that this should lead to a positive, indirect effect of howler monkey density on bird species richness. After accounting for passive sampling (the tendency for species richness to be positively associated with island area, regardless of differences in habitat quality) we found a significant positive correlation between howler monkey density and bird species richness. A path analysis incorporating data on tree growth rates from a subset of islands (n = 9) supported the hypothesis that the effect of howler monkeys on the resident bird communities is indirect and is mediated through changes in plant productivity and habitat quality. These results highlight the potential for disparate taxonomic groups to be related through indirect interactions and trophic cascades.     

Vigilance and mixed-species association of some East African forest monkeys

Summary The frequency of looking up was scored as a measure of vigilance behavior in two species of African forest monkeys (genus Cercopithecus) that often associate in mixed-species groups. The fact that looking up decreased with increasing foliage density around focal individuals was taken as evidence that looking up is an expression of vigilance for predators. The rate of looking up was higher in single-species groups than in mixed species groups for both species. Association had a more marked effect on the rate of looking up when monkeys fed on plant material as opposed to insects. The adjustment of the rate of looking up with respect to association status does not reflect the presence or absence of other monkeys in the same feeding tree. Although a reduction in vigilance levels probably leads to increased feeding efficiency, it is not a sufficient explanation of mixed-species association in the species under study.     

Dark, bitter-tasting nectar functions as a filter of flower visitors in a bird-pollinated plant

Floral nectar is offered by plants to animals as a reward for pollination. While nectar is typically a clear liquid containing sugar and trace amounts of amino acids, colored nectar has evolved in several plant families. Here we explore the functional significance of the phenolic compounds that impart a dark brown color to the nectar of the South African succulent shrub Aloe vryheidensis. Flowers of this aloe are visited for their nectar by a suite of short-billed birds that are occasional nectarivores, including bulbuls, white-eyes, rock thrushes, and chats. Dark-capped Bulbuls were more likely to probe model flowers containing dark nectar than those containing clear nectar, suggesting a potential signaling function for dark nectar. However, the main effect of the phenolics appears to be to repel "unwanted" nectarivores that find their bitter taste unpalatable. Nectar-feeding honey bees and sunbirds are morphologically mismatched for pollinating A. vryheidensis flowers and strongly reject its nectar. However, the frugivorous and insectivorous birds that effectively pollinate this aloe are seemingly unaffected by the nectar's bitter taste. Thus the dark phenolic component of the nectar appears to function as a floral filter by attracting some animals visually and deterring others by its taste.     

Work division of floral scent compounds in mediating pollinator behaviours

Floral scents are known as an olfactory signal for attracting pollinators, but why the flowers pollinated by highly specialised pollinators emit scents consisting of mixtures of many compounds and dominated by one or a few compounds is still poorly understood. We supposed that each (especially characteristic) chemical in floral scents may play a specific role in mediating pollinator behaviours and tested this supposition in a fig-fig wasp mutualism. Ficus curtipes is obligately pollinated by an undescribed Eupristina species. In the scent of F. curtipes receptive figs, over 50 compounds have been identified, and the scent is dominated by two compounds, 6-methyl-5-hepten-2-ol (OL) and 6-methyl-5-hepten-2-one (NE). We therefore tested the roles of the two major chemicals in mediating the pollinator behaviours. Our results show that OL and NE, respectively, act as a long-distance attractant and a fig-entry behaviour stimulant to the obligate pollinator wasp. Namely, OL attracts the wasps to the figs and NE guides the wasps into the figs. This finding on the work division of floral scent compounds partially explains the maintenance mechanism of the fig-fig wasp mutualism and the significance of the chemical diversity of floral scent in plant–pollinator interactions, especially in specialised pollination systems.     

Effects of Native and Non‐Native Vertebrate Mutualists on Plants

Abstract: Extinctions can leave species without mutualist partners and thus potentially reduce their fitness. In cases where non‐native species function as mutualists, mutualism disruption associated with species’ extinction may be mitigated. To assess the effectiveness of mutualist species with different origins, we conducted a meta‐analysis in which we compared the effectiveness of pollination and seed‐dispersal functions of native and non‐native vertebrates. We used data from 40 studies in which a total of 34 non‐native vertebrate mutualists in 20 geographic locations were examined. For each plant species, opportunistic non‐native vertebrate pollinators were generally less effective mutualists than native pollinators. When native mutualists had been extirpated, however, plant seed set and seedling performance appeared elevated in the presence of non‐native mutualists, although non‐native mutualists had a negative overall effect on seed germination. These results suggest native mutualists may not be easily replaced. In some systems researchers propose taxon substitution or the deliberate introduction of non‐native vertebrate mutualists to reestablish mutualist functions such as pollination and seed dispersal and to rescue native species from extinction. Our results also suggest that in places where all native mutualists are extinct, careful taxon substitution may benefit native plants at some life stages.