Mixed Breeding System Research Articles

The potential role of two LEAFY orthologs in the chasmogamous/cleistogamous mixed breeding system ofViola pubescens(Violaceae)1

Abstract Many plants, including most species in Viola, produce both open, outcrossing chasmogamous and closed, self-pollinating cleistogamous flowers. The chasmogamous/cleistogamous mixed breeding system is considered an evolutionarily successful reproductive strategy, but the underlying molecular basis remains largely unknown. The LEAFY (LFY) gene in Arabidopsis is responsible for the initiation of floral meristems and the regulation of flower development; therefore, we sought to identify LFY orthologs in Viola pubescens Aiton with the goal of understanding possible differences in the regulatory genetics of flower development and the molecular genetics underlying chasmogamous/cleistogamous mixed breeding systems. A genomic library of V. pubescens was constructed to identify LFY orthologs, and reverse transcription polymerase chain reaction was employed to study gene expression in both flower types. In addition, overexpression studies of the orthologs were carried out in Arabidopsis to explore their role ...

Development and characterization of microsatellite loci for the Neotropical orchid Trichocentrum pumilum

Abstract Studies of genetic diversity and structure are key elements in designing effective in situ and ex situ management plans, especially for species experiencing forest fragmentation. To investigate the level of genetic diversity in populations of Trichocentrum pumilum, eight polymorphic microsatellite loci were developed and used for genotyping 96 specimens from four disturbed populations. Low genetic diversity within populations was found (average number of alleles per locus ranging from 3.75 to 4.25, observed and expected heterozygosities from 0.238 to 0.333 and from 0.450 to 0.482, respectively). The fixation index () ranged from 0.35 to 0.47, with significant values for all populations. No genotypic disequilibrium was detected. A mixed breeding system was found through an apparent outcrossing rate estimate. Our results suggest that these microsatellite loci are suitable for genetic studies of this species, showing low within population genetic diversity and moderate structure for T. pumilum populations.

Reproductive Biology of Mangrove PlantsClerodendrum inerme, Derris trifoliata, Suaeda maritima, Suaeda monoica, Suaeda nudiflora

AbstractFloral biology, sexual system, breeding system, pollinators, fruiting and seed dispersal aspects of five mangrove associate species, namely,Clerodendrum inerme,Derris trifoliata,Suaeda maritima, S. monoica, andS. nudiflorawere studied. All these species are hermaphroditic, self-compatible, and exhibit mixed breeding systems adapted for pollination by external agents.C. inermeandD. trifoliataare principally entomophilous, whileSuaedaspecies are ambophilous. The study suggests that these plant species are important constituents of mangrove forests.C. inermeandD. trifoliataare useful in stabilizing the banks of back water creeks, whileSuaedaspecies are useful to carpet the saline areas and desalinate the soils which are subsequently useful for agricultural activities.

On the reproductive ecology of Suaeda maritima, S. monoica and S. nudiflora (Chenopodiaceae).

Floral biology, sexual system, breeding system, pollinators, fruiting and seed dispersal aspects of three Suaeda species, S. maritima, S. monoica and S. nudiflora (Chenopodiaceae) were studied. The flowers of all the three species are hermaphroditic, dichogamous, strongly protogynous with a pistillage phase during the mature bud stage and staminate phase following anthesis, self-compatible exhibiting mixed breeding systems with special adaptation for cross-pollination; but both self- and cross-pollination are vector-dependent. In all, the flowers display a mix of anemophilous and entomophilous traits. Anemophily is effective in high salt marshes while water currents bring about pollination in low salt marshes; insects pollinate the flowers while collecting the forage from pistillate and staminate phase flowers. In these species, the whole plant breaks off and rolls on the floor while shedding its diaspores. Fruits with seeds intact and/or seeds shed from fruits float on water due to their ability for buoyancy. The fruits and seeds thus disperse, settle in the entire extent of salt marshes or coastal areas and germinate in mid-summer season when salinity is very high in high and low salt marshes.

Pollination ecology of Derris trifoliata (Fabaceae), a mangrove associate in Coringa Mangrove Forest, Andhra Pradesh, India

Derris trifoliata is a perennial woody climber. It blooms massively for about two weeks in July/August. The flowers are hermaphroditic, feebly protandrous, self-compatible and display a vector dependent mixed breeding system. They close back by the end of the day of anthesis. The forenoon anthesis and pollen and nectar as rewards attract daytime foragers. The nectar feeding foragers require strength to depress the keel petals in order to collect nectar; only those foragers which have the required strength to do so can collect nectar and in the process trip the floral mechanism and effect pollination. When floral explosion occurs, the pollen is somewhat exposed and the pollen feeding foragers then collect it. Both long- and short-tongued bees trip the flowers, collect nectar and effect pollination. Individual flowers that were not tripped by insects set fruit to negligible level. In open-pollination mode, fruit set rate is up to 30-31% only despite the flowers being visited by insect pollinators. Fruits mature quickly within a month. Each fruit contains 1-3 seeds against 6 linearly arranged ovules in the ovary. The fruits are leathery and possess air cavities, the characteristics of which enable them to float in tidal water. They settle at the parent plant if the site is partly or fully exposed or float for dispersal if the site is inundated with tidal water. Seed release occurs when fruits absorb water and the pericarp breaks. Seeds germinate only when they reach a suitable habitat in mangroves.

Pollination ecology of Clerodendrum inerme (L.) Gaertn. (Lamiaceae) in Coringa mangrove ecosystem, Andhra Pradesh, India

Clerodendrum inerme (L.) Gaertn. (Lamiaceae) is bisexual, self-compatible and has a vector-dependent mixed breeding system. They are dichogamous and herkogamous; the day 1 flowers are staminate while the day 2 and 3 flowers are pistillate. The plant blooms in the evening, possesses a white long corolla with a hairy interior to exclude other insects and strong fragrance are adaptations for pollination by the hawk-moth Macroglossum gyrans. The 2nd and 3rd day flowers are nectar-rich and attract hawk-moths during the dawn and dusk hours. The plant is also visited by bees and butterflies. The bees Xylocopa and Anthophora are primary nectar robbers which collect nectar without effecting pollination. In C. inerme, three forms of flowers can be distinguished based on the position of sex organs. The first form is characterized by elongated stamens and a style which occur in close proximity to each other just after anthesis facilitating contact between the stamens and stigma. The second form is characterized by the scattered position of stamens and style. In the third form, the stamens are fully extended while the style is curved away from them, either to the left or to the right; subsequently the stamens curl inward and the style elongates. Interestingly, the three flower forms can be found within a cyme also. These forms of flowers with strong protandry prevent autonomous selfing but not geitonogamy. The fruit is a capsule and breaks open to disperse nutlets. Birds such as Acridotheres tristis, Corvus splendens, Corvus macrorhynchos and Turdoides caudatus disperse nutlets during the early winter season. Seeds germinate in June and seedlings grow gradually to produce new plants.

Breeding system, shell size and age at sexual maturity affect sperm length in stylommatophoran gastropods.

BackgroundSperm size and quality are key factors for fertilization success. There is increasing empirical evidence demonstrating that sperm form and function are influenced by selective pressures. Theoretical models predict that sperm competition could favour the evolution of longer sperm. In hermaphrodites, self-fertilizing species are expected to have shorter sperm than cross-fertilizing species, which use sperm stored from several mating partners for the fertilization of their eggs and thus are exposed to intense sperm competition. We tested this hypothesis by comparing original data on sperm length in 57 species of simultaneously hermaphroditic stylommatophoran gastropods from Europe and South America with respect to the species’ breeding system. We used 28S rRNA nuclear and COI mitochondrial sequence data to construct a molecular phylogeny. Phylogenetic generalized linear models were applied to examine the potential influence of morphological and life-history characters.ResultsThe best-fit model revealed that the breeding system and age at sexual maturity influence sperm length in gastropods. In general, species with predominant cross-fertilization had longer sperm than species with predominant self-fertilization or a mixed breeding system. Across species with shells (snails), sperm length also increased with shell size.ConclusionsOur study provides evidence that sperm length in stylommatophoran gastropods is influenced by the risk of sperm competition, as well as by age at sexual maturity and shell size. This finding extends present knowledge of sperm evolution to a group of so far poorly studied simultaneous hermaphrodites.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0661-9) contains supplementary material, which is available to authorized users.

All in an afternoon: mixed breeding system in one-day lasting flowers of Hypericum elodes L. (Hypericaceae)

Knowledge about mixed mating systems can improve our understanding of the evolutionary dynamics of reproductive systems. Here we report a study of the floral and reproductive biology of Hypericum elodes, an Atlantic-European soft-water pools specialist which shows a floral architecture consistent with both self- and cross-pollination. Controlled pollination experiments were performed in a natural population during three consecutive years. Marked flowers were monitored until fruit production, and laboratory germination experiments were conducted with the seeds produced. Plants were self-compatible (SCI>0.75), however, compared with selfing, cross-pollination enhanced fruit-set, seed-set and seedling growth, but not seed germination. Inbreeding depression (δ) was mild in the pre-dispersal stages (δ = 0.22 for fruit set, 0.18 for seed set and 0.13 for seed mass), low for germination percentage (δ = 0.003) and mild for seedling growth (δ = 0.23). The breeding system of H. elodes promotes outcrossing and assures reproductive success by means of competitive autogamy. Our results suggest a mixed mating strategy for the studied population, characterized by mild inbreeding depression (cumulative δ = 0.57), highlighting the benefit of this reproductive mode in unpredictable habitat, as the typical shallow-water meadows where H. elodes grows.

Reproduction and Pollination of the Endangered Dwarf Bear-PoppyArctomecon humilis(Papaveraceae) across a Quarter Century: Unraveling of a Pollination Web?

Arctomecon humilis, a rare gypsophile of the extreme northeastern Mojave Desert, is restricted to a few isolated populations in Washington County, Utah, USA. At several times in the past quarter century, we have studied the breeding system and reproductive success of this endangered species, recorded its pollinators, and tested the feasibility of human-assisted gene flow by performing reciprocal crosses between 2 isolated “populations” approximately 4 km apart. Arctomecon humilis possessed a mixed breeding system in the population studied (Beehive Dome in 1988); some plants exhibited self-compatibility but produced significantly fewer fruit/flowers and seeds/fruit for geitonogamous self-pollinations than for cross-pollinations. Few fruits and seeds were produced in the absence of pollinators. The results of cross-pollination treatments did not differ from unassisted open-pollinations (controls), suggesting that pollinators were not limiting reproductive success. Our more recent results (2012) suggest that some populations may be more at risk than others. Although all 7 populations surveyed produced ≥70% fruits/flower, they differed significantly in fruit set and in average seed number/fruit and seed weight. Possible reasons for these differences (i.e., inbreeding, genetic load, insufficient mating types, pollinator scarcity, etc.), are all potentially important for conservation management and should be investigated. Over the past 2 decades, the pollinator community appears to have changed dramatically from one composed of specialist and generalist bees to one where pollination is presently being accomplished by generalist foragers alone. The health of one of these generalists, the honey bee Apis mellifera, is a current global concern, and its future presence as a pollinator of A. humilis is unclear. Our reciprocal crosses between the White Dome and Webb Hill populations provide support for our proposal that human-mediated gene flow through interpopulation cross-pollinations be undertaken every 5 years to increase the genetic variability of populations. We make several other recommendations for research that would improve the ability of land managers to conserve this species.

Pollination ecology of the Gray Nicker Caesalpinia crista (Caesalpiniaceae) a mangrove associate at Coringa Mangrove Forest, Andhra Pradesh, India

Caesalpinia crista L., commanly known as Gray Nicker, is an oligohaline mangrove associate confined to landward marginal areas of the Coringa Mangrove Forest, Andhra Pradesh, India. The flowering occurs during the wet season from June to November. The flowers are hermaphroditic, self-compatible and exhibit a mixed breeding system. The floral characteristics that constitute melittophilous pollination syndrome include diurnal anthesis, slight fragrance, zygomorphy, yellow petals, with a flag petal displaying a conspicuous nectar guide, and the presence of nectar with a high sugar concentration. Extra-floral nectar along the rachis is an additional attractant and is easily perceivable by bees. The plant is pollinated almost exclusively by bees, especially carpenter bees. The floral characteristics such as free petals, fully exposed stamens with dry and powdery pollen grains and hairy stigma facilitate anemophily which is effective due to high winds during the rainy season. The prolific growth and near synchronous flowering at population level contribute to pollen availability in huge quantities and enable anemophily as an effective mode of pollination. The functionality of melittophily and anemophily together constitutes ambophily. Hand-pollination experiments indicated that the plant is principally out-crossing. The natural fruit set does not exceed 10%; this lowest percentage could be partly due to flower-feeding by the beetle, Mylabris phalerata. The fruits are indehiscent, 1-seeded, which are buoyant and are not dispersed far away from the parental sites. The viable seeds produce new plants in the vicinity of parental plants during the rainy season. This plant builds up its population as small patches or in pure stands and hence is important in building landward mangrove cover.

Effects of flower dimorphism and light environment on arbuscular mycorrhizal colonisation in a cleistogamous herb.

Although it is known that floral dimorphism contributes to the maintenance of mixed breeding systems, the consequences of producing progeny of a contrasting genetic background and seeds with differential resource allocation has been practically ignored regarding establishment of belowground organisms-plant interactions. This article evaluates the combined effect of floral dimorphism with cross type and light environment on interactions between Ruellia nudiflora and arbuscular mycorrhizal fungi (AMF). R.nudiflora produces cleistogamous (CL) flowers that exhibit obligate self-pollination and chasmogamous (CH) flowers with facultative self- (CHs) or cross- (CHc) pollination. We evaluated the establishment of the plant-AMF interaction in progeny derived from each floral type, under two light conditions (shaded versus open). We established different scenarios depending on the existence of inbreeding depression (ID) and whether the differential resource allocation (DRA) to CH and CL flowers affected the R.nudiflora-AMF interaction. We predicted that under shaded light conditions there might be an intensification of ID, having a negative effect on AMF colonisation. The percentages of hyphae and vesicles in the harvested roots was significantly higher in the shaded plants (F≥4.11, P<0.05), while progeny of CHc and CHs presented a higher percentage of hyphae and vesicle colonisation compared to CL progeny (F=15.26, P<0.01). The results show that DRA to CH flowers and light availability both determines the establishment of R.nudiflora-AMF interaction. The results also suggest that even under stressful light conditions, endogamy does not affect this interaction, which may explain the success of R.nudiflora as an invasive species.

Naturalization and invasion of alien plants in Puerto Rico and the Virgin Islands

A fundamental goal in invasion ecology is to identify and understand the factors explaining why some alien species become invasive when others fail. In this study we gathered data on taxonomy, invasive status, invasion history, geographic distribution, and biological and ecological traits of 1,032 alien plant species occurring on different habitats in Puerto Rico and the Virgin Islands. These data were used to evaluate the relative importance of habitat and species attributes in influencing the likelihood of alien plants to become naturalized and subsequently invasive on these islands. Our results showed that alien species were more likely to become naturalized if they grow in semi-natural habitats, have large native distribution ranges, long residence time, were introduced for horticulture and have mixed breeding systems. On the other hand, naturalized species were more likely to become invasive if they were introduced for horticul- ture, behave as weeds, have tolerance to different climatic life-zones, thrive in ruderal habitats, have mixed breeding systems with hermaphroditic flowers, reproduce vegetatively and produce small seeds. Whereas some of these parameters were important for both transitions, others were important for either naturalization or invasion. Overall, our results empha- size the importance of studying different stages of the invasion process in order to understand the mecha- nisms explaining successful invasions rather than baseline approaches of simply comparing invasive with natives or noninvasive alien species. Understand- ing the process by which alien species become naturalized or invasive could provide a more objective and accurate approach for managing and predicting biological invasions.

MIXED BREEDING SYSTEM AND ENTOMOPHILY IN Malachra capitata L. (MALVACEAE)

Malachra capitata is a small seasonal herb. Flowering occurs during September–October. It is hermaphroditic and self-compatible with a mixed breeding system. The conspicuous floral displays, non-tubular nature of the flower, and the small volume of nectar with high sugar concentration attract bees and butterflies with bees serving as the principal pollinator. The fruit set is 21% in open-pollination mode despite the hectic foraging activity of bees and butterflies. The observed fruit set rate is attributed to the high percentage of flower predation by beetles, and by the nature of soil nutrient/environment requirements. The fruit is a schizocarp with five 1-seeded indehiscent mericarps. New plants arise from seed during the rainy season mostly at parental sites and usually form pure stands; the plants complete their life cycle within six or seven months.

Mixed breeding system in the hermaphroditic land slugArion intermedius(Stylommatophora, Arionidae)

Theory suggests that hermaphroditic plants and animals should be either entirely outcrossing or entirely selfing. As such, very few hermaphroditic plants and basommatophoran snails have a mixed breeding system. However, reliable estimates of selfing rates are lacking for most hermaphroditic animals. This partly prevents to delineate the relative contributions of the selective factors that determine selfing and outcrossing rates in hermaphroditic animal taxa. Here, we studied the population genetic structure of, and breeding system in, 11 populations of the hermaphroditic land slug Arion intermedius using five polymorphic microsatellite loci. Moreover, genotype frequencies deviated significantly from Hardy-Weinberg equilibrium expectations for most of the loci in all populations suggesting some level of selfing. Estimates of the selfing level s, suggest moderate levels of outcrossing (mean s based on FIS = 0.84; mean s based on the two-locus heterozygosity disequilibrium = 0.20, or with a ML approach = 0.22). Our study therefore suggests that A. intermedius has a mixed breeding system. A re-analysis of allozyme data from another arionid slug ( subgenus Carinarion) indicates that mixed breeding may be more common in arionid slugs than hitherto was assumed. These results seem therefore at variance with current theoretical and empirical predictions and opens perspectives for the study on the evolutionary factors driving mixed breeding systems in animals.

The genetic basis and experimental evolution of inbreeding depression in Caenorhabditis elegans

Determining the genetic basis of inbreeding depression is important for understanding the role of selection in the evolution of mixed breeding systems. Here, we investigate how androdioecy (a breeding system characterized by partial selfing and outcrossing) and dioecy (characterized by obligatory outcrossing) influence the experimental evolution of inbreeding depression in Caenorhabditis elegans. We derived inbred lines from ancestral and evolved populations and found that the dioecious lineages underwent more extinction than androdioecious lineages. For both breeding systems, however, there was selection during inbreeding because the diversity patterns of 337 single-nucleotide polymorphisms (SNPs) among surviving inbred lines deviated from neutral expectations. In parallel, we also followed the evolution of embryo to adult viability, which revealed similar starting levels of inbreeding depression in both breeding systems, but also outbreeding depression. Under androdioecy, diversity at a neutral subset of 134 SNPs correlated well with the viability trajectories, showing that the population genetic structure imposed by partial selfing affected the opportunity for different forms of selection. Our findings suggest that the interplay between the disruptions of coevolved sets of loci by outcrossing, the efficient purging of deleterious recessive alleles with selfing and overdominant selection with outcrossing can help explain mixed breeding systems.

Taxonomic and population genetic re-interpretation of two color morphs of the decollate snail, Rumina decollata (Mollusca, Pulmonata) in southern France

The hermaphroditic terrestrial snail Rumina decollata has a mixed breeding system with a high prevalence of self-fertilization. In the Montpellier area (France), the species is represented by a dark and a light color morph. Based on allozyme data, both morphs have been reported as single, homozygous multilocus genotypes (MLG), differing at 13 out of 26 loci, but still showing occasional hybridization. Recent DNA sequence data suggest that each morph is a different phylogenetic species. In order to further evaluate this new taxonomic interpretation, the present contribution explores to what extent populations or color morphs indeed consist of single or few MLG. As such it is shown that both morphs are not single, homozygous MLG, but instead reveal a considerable amount of allelic variation and substantial numbers of heterozygous microsatellite genotypes. This suggests that outcrossing may be more prevalent than previously reported. Nevertheless, both morphs maintain a diagnostic multimarker differentiation in the presence of outcrossing in sympatric conditions, implying that they may be interpreted as species under the biological species concept. Finally, our data challenge the idea that simultaneous hermaphrodites should be either strict selfers or strict outcrossers.

POLLINATION ECOLOGY OF Lumnitzera racemosa WILLD. (COMBRETACEAE), A NON-VIVIPAROUS MANGROVE TREE

Lumnitzera racemosa is a non-viviparous evergreen mangrove tree species. It is a massive bloomer from mid-July to mid-August. The flowers are bisexual, self-compatible, self-pollinating and exhibit a mixed breeding system. The plant is temporally dioecious with marked protandry and a non-receptive stigma on day one of anthesis with a receptive stigma on days two and three. Fruit set is approximately 90% due to self and cross-pollination. The floral characters are related to pollination by generalized flower-visiting insects, notably actinomorphy, white spreading petals, limited but easily accessible nectar, and position of stamens. Pollinators include bees, wasps and butterflies. Fruits are invariably 1-seeded despite the presence of 3–5 ovules. Natural regeneration is low perhaps due to abortion of embryos or the feeding of embryos by small grubs. Fallen mature fruits become fibrous in water and this may aid dispersal in the sea. However, fruit germination and seedling establishment is curtailed due to their salinity-sensitivity.

Exploring species level taxonomy and species delimitation methods in the facultatively self-fertilizing land snail genus Rumina (gastropoda: pulmonata).

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.

Gibberellins are involved but not sufficient to trigger a shift between chasmogamous-cleistogamous flower types in Viola pubescens1

Abstract At least 50 angiosperm families have plants that produce both chasmogamous flowers and cleistogamous flowers. Various environmental and physiological factors, including the plant growth regulators gibberellins (GAs), have been reported to influence the flower types. Here, the relationship between GAs and flower production was studied for the first time in Viola, a genus famous for the large number of species with the mixed breeding system. Orthologs of genes for GA20 oxidase (VGA20ox) and GA3 oxidase (VGA3ox) were identified by reverse transcription polymerase chain reaction (RT-PCR) from a widespread North American species, Viola pubescens. Semi-quantitative RT-PCR indicated that both genes had increased expression in chasmogamous flowers as compared to cleistogamous flowers, supporting a role for GA in the differential production of flower type. However, the application of exogenous GA3 (the most common commercially available GA) to V. pubescens failed to induce a conversion of production of cl...

The reproductive biology of island plants: the Juan Fernández and Canary Islands

Knowledge of reproductive biology is fundamental to understanding successful colonization and subsequent evolution of island plants, to founding effective conservation programs for island biota, and for the insights provided from island studies to understanding the evolution of plants in general. We focus on the reproductive biology of plants of the Canary Islands via a comparison with that of plants from the Chilean Juan Fernández (Robinson Crusoe Islands). We focus on breeding systems (i.e., compatibility, ability to self pollinate) and pollination. The breeding system of the progenitors of island lineages are inferred to assess the usual interpretation: i.e., that successful colonists are self compatible, i.e., following ‘Baker’s Law’ vs. the alternative of a mixed breeding system (‘leaky’ self incompatibility). We briefly review the mechanisms promoting outcrossing in self compatible hermaphroditic species. We assess whether current floral forms reflect pollination features of the colonizing ancestors or whether they represent selection on islands in situ. . The limited range of pollinators available to new island colonists especially of younger archipelagoes, forces adaptation to new syndromes that are not always reflected in their floral morphology or nectar. Despite the broad interest in island plants, there are few comprehensive studies of either breeding systems or pollination biology. Thus, generalizations about island systems can lead to misunderstandings: as Baker exhorted nearly a half century ago, more studies of island plant reproductive biology are needed. Furthermore, conservation programs will not be effective without a knowledge of reproductive biology of island natives.