Migration load and the coexistence of ecologically similar sexuals and asexuals

The frozen niche variation hypothesis suggests that sexuals can coexist with closely related, ecologically similar asexuals because sexuals and narrowly adapted asexual clones use different resources. However, because a collection of clones can potentially dominate the entire resource axis, such coexistence is not stable. We show that if the sexual population inhabits multiple selection regimes and asexuals are intrinsically slightly less fit than sexuals, migration load in the sexual population allows sexuals and asexuals to coexist stably at the regional level. By decreasing sexuals' fitness, migration load allows asexuals to invade the sexual population. However, as the sexuals' range contracts, migration load decreases, preventing asexuals from driving sexuals to extinction. This "buffering" effect of migration load is even more relevant in models that include more realistic conditions, such as demographic asymmetries or explicit spatial structure.     

Making it on their own: sperm-dependent hybrid fishes (Cobitis) switch the sexual hosts and expand beyond the ranges of their original sperm donors

Interspecific hybridization may result in asexual hybrid lineages that reproduce via parthenogenesis. Contrary to true parthenogens, sperm-dependent asexuals (gynogens and hybridogens) are restricted to the range of bisexual species, generally the parental taxa, by their need for a sperm donor. It has been documented that asexual lineages may rarely use sperm from a non-parental species or even switch a host. The available literature reports do not allow distinguishing, between whether such host switches arise by the expansion of asexuals out of their parental's range (and into that of another's) or by the local extinction of a parental population followed by a host switch. The present study combines new and previously collected data on the distribution and history of gynogenetic spined loaches (Cobitis) of hybrid origin. We identified at least three clonal lineages that have independently switched their sperm dependency to different non-parental Cobitis species, and in cases incorporated their genomes. Our current knowledge of European Cobitis species and their hybrids suggests that this pattern most probably results from the expansion of gynogenetic lineages into new areas. Such expansion was independent of the original parental species. This suggests that sperm dependence is not as restrictive to geographical expansion when compared with true parthenogenesis as previously thought.     

Coexistence of sperm-dependent asexuals and their sexual hosts: the role of differences in fitness-related traits

Understanding the mechanisms of coexistence of sperm-dependent asexuals and their sexual hosts requires an empirical evaluation of their relative fitness. We evaluated the fecundity, egg viability, and hatchling growth rate of the sperm-dependent asexual hybrid species Phoxinus eos-neogaeus and its sexually-reproducing parental species, P. eos and P. neogaeus. Three comparisons between asexual and sexual Phoxinus suggested a fitness advantage in the asexuals: absolute fecundity was higher in P. eos-neogaeus than P. eos, but this advantage disappeared when fecundity differences were scaled to differences in body size; a higher fraction of P. eos-neogaeus eggs than P. eos eggs were viable post-fertilization; P. eos-neogaeus grew faster in the first 60 days post-hatching than both their P. eos and P. neogaeus hosts. All other comparisons showed no difference in fitness between P. eos-neogaeus and the parental species. Overall, our data suggest, at best, a weak fitness advantage for the asexuals, which suggests that other factors, such as niche separation or male preference for sexually-reproducing females during mating, are critical to the maintenance of asexual-sexual lineages.     

Role of parasite load and differential habitat preferences in maintaining the coexistence of sexual and asexual competitors in fish of the Cobitis taenia hybrid complex

In the context of the paradoxical ubiquity of sex, we tested whether stable coexistence of sexual and asexual fish of the genus Cobitis is mediated by parasites, as asexual fish suffer more from parasitic infections because of their lower genetic variability [the Red Queen hypothesis (RQH)], or by partial niche shift of the two strains differing in mode of reproduction. We did not find a clear correlation between infection risk with a helminth parasite and the proportion of sexuals, and we found similar infection rates among sexual females and co‐occurring asexuals in general, including the most frequent clone in particular. These results suggest that the mechanisms of the RQH are not directly engaged in stabilizing this asexual complex. On the other hand, the temporally stable gradient in sexual/asexual proportions along the river correlated with gradients in environmental parameters (physicochemical water parameters, velocity, and shading of the habitat) and turnover in the fish assemblage structure. Sexual and asexual forms thus appear to prefer different habitats. The Cobitis teania asexual complex thus contributes to the view that persistence of sex may, as in many taxa, be driven by case‐specific processes. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 220–235.     

The effect of rearing temperature on body shape and meristic characters in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) juveniles

Considering its immediate costs of producing dispensable males, the maintenance of sexual reproduction is a major paradox in evolutionary biology. Asexual lineages that do not face such costs theoretically should replace sexuals over time. Nonetheless, several systems are known in which closely related sexual and asexual lineages stably coexist. In the present study, we studied a sexual/asexual mating complex of a sperm-dependent parthenogenetic fish (amazon molly, Poecilia formosa) and its sexual congeners, the sailfin molly P. latipinna and the Atlantic molly P. mexicana. We asked whether differences in feeding behavior could contribute to their stable coexistence. We conducted a laboratory experiment to compare feeding efficiencies and also measured the competitive abilities between the two reproductive forms. Additionally, we measured gut fullness of fishes caught in natural habitats. Contrary to our predictions, we could not find P. formosa to be less efficient in feeding. We argue that food competition in mollies plays a minor role in mediating coexistence between closely related asexual and sexual mollies.     

Female sperm limitation in natural populations of a sexual/asexual mating complex (Poecilia latipinna, Poecilia formosa)

In sperm-dependent sexual/asexual mating systems, male mate choice is critical for understanding the mechanisms behind apparent stability observed in natural populations. The gynogenetic Amazon molly (Poecilia formosa) requires sperm from sexual males (e.g. Poecilia latipinna) to trigger embryogenesis, but inheritance is strictly maternal. Consequently, males should try to avoid or reduce the cost of mating with asexuals. We investigated male mate choice by documenting the presence of sperm in natural populations and found that a higher proportion of sexual females had sperm than asexuals. In addition, among those females that had sperm, sexuals had more sperm than asexuals. Our results hint at a role for male mate choice as a stabilizing factor in such systems.     

Discordance between nuclear and mitochondrial genomes in sexual and asexual lineages of the freshwater snail Potamopyrgus antipodarum

The presence and extent of mitonuclear discordance in coexisting sexual and asexual lineages provides insight into 1) how and when asexual lineages emerged, and 2) the spatial and temporal scales at which the ecological and evolutionary processes influencing the evolution of sexual and asexual reproduction occur. Here, we used nuclear single‐nucleotide polymorphism (SNP) markers and a mitochondrial gene to characterize phylogeographic structure and the extent of mitonuclear discordance in Potamopyrgus antipodarum. This New Zealand freshwater snail is often used to study the evolution and maintenance of sex because obligately sexual and obligately asexual individuals often coexist. While our data indicate that sexual and asexual P. antipodarum sampled from the same lake population are often genetically similar, suggesting recent origin of these asexuals from sympatric sexual P. antipodarum, we also found significantly more population structure in sexuals vs. asexuals. This latter result suggests that some asexual lineages originated in other lakes and/or in the relatively distant past. When comparing mitochondrial and nuclear population genetic structure, we discovered that one mitochondrial haplotype (‘1A’) was rare in sexuals, but common and widespread in asexuals. Haplotype 1A frequency and nuclear genetic diversity were not associated, suggesting that the commonness of this haplotype cannot be attributed entirely to genetic drift and pointing instead to a role for selection.     

VARIATION IN THE STRENGTH OF MALE MATE CHOICE ALLOWS LONG‐TERM COEXISTENCE OF SPERM‐DEPENDENT ASEXUALS AND THEIR SEXUAL HOSTS

In several asexual taxa, reproduction requires mating with related sexual species to stimulate egg development, even though genetic material is not incorporated from the sexuals (gynogenesis). In cases in which gynogens do not invest in male function, they can potentially have a twofold competitive advantage over sexuals because the asexuals avoid the cost of producing males. If unmitigated, however, the competitive success of the asexuals would ultimately lead to their own demise, following the extinction of the sexual species that stimulate egg development. We have studied a model of mate choice among sexual individuals and asexual gynogens, where males of the sexual species preferentially mate with sexual females over gynogenetic females, to determine if such mating preferences can stably maintain both gynogenetic and sexual individuals within a community. Our model shows that stable coexistence of gynogens and their sexual hosts can occur when there is variation among males in the degree of preference for mating with sexual females and when pickier males pay a higher cost of preference.     

How to go extinct by mating too much: population consequences of male mate choice and efficiency in a sexual–asexual species complex

Selection acting on individuals is not predicted to maximize population persistence, yet examples that explicitly quantify conflicts between individual and population level benefits are scarce. One such conflict occurs over sexual reproduction because of the cost of sex: sexual populations that suffer the cost of producing males have only half the growth rate compared to asexuals. Male behaviour can additionally impact population dynamics in a variety of ways, and here we study an example where the impact is unusually clear: the riddle of persistence of sperm‐dependent sexual–asexual species complexes. Here, a sexually reproducing host species coexists with an ameiotically reproducing all‐female sperm parasite. Sexual–asexual coexistence should not be stable because the proportion of asexually reproducing females will rapidly increase and the relative abundance of the sexually reproducing host species will decline. A severe shortage of males will lead to sperm limitation for sexual and asexual females and the system collapses. Male mate choice could reduce the reproductive potential of the asexual species and thus potentially prevent the collapse. In the gynogenetic (sperm‐dependent parthenogenetic) Amazon molly Poecilia formosa and its host (P. latipinna or P. mexicana), males discriminate against asexual females to some extent. Using a population‐dynamical model, we examine the population dynamics of this species complex with varying strengths of male discrimination ability and efficiency with which they locate females and produce sperm. The sexual species would benefit from stronger discrimination, thus preventing being displaced by the asexual females. However, males would be required to evolve preferences that are probably too strong to be purely based upon selection acting on individuals. We conclude that male behaviour does not fully prevent but delays extinction, yet this is highly relevant because low local extinction rates strongly promote coexistence as a metapopulation.     

THE DISTRIBUTION OF LIFE-HISTORY VARIATION IN THE DAPHNIA PULEX COMPLEX

In the midwestern United States the Daphnia pulex complex consists of a mosaic of sexual and asexual populations, providing a useful model system for studying the evolutionary forces underlying the maintenance of sex. One asexual and two sexual populations were surveyed for genetic variation for isozymes, mitochondrial DNA, and life-history characters. While the sexual populations exhibited substantial levels of genetic variance for fitness characters, no variation was detected in the asexual population at any level. However, a parallel survey among asexual clones derived from other ponds revealed large amounts of quantitative variation among clones, even among those with the same molecular profile. As a group, the asexuals are more variable for life histories than are the sexual populations. The molecular data indicate a relatively recent origin for the extant asexual D. pulex. The polyphyletic origin of these clones, combined with their microevolutionary potential, provides an explanation for their broad geographic distribution. The distribution of sex in the complex cannot be explained with the standard models that assume an invariant asexual population in reproductive isolation from the parental species. Although the frequency of asexuality may be driven by the spread of a sex-limited meiosis suppressor through sexual populations, the complete displacement of sexuality may be prevented by ecological distinctions between the two classes of individuals. On average, the asexuals are larger but produce smaller clutches than the sexuals.     

Juvenile survival in a unisexual/sexual complex of mollies

Unisexual species like the gynogenetic Amazon molly, Poecilia formosa, enjoy a twofold advantage over sexual species, because they do not produce males. Therefore, unisexuals should be able to outcompete and consequently, replace sexual species. For sperm-dependent (gynogenetic) unisexuals this creates a paradox: they cannot replace their sexual hosts without eradicating themselves. Thus, mechanisms must be in place to stabilize such mating systems. We assessed juvenile survivorship between asexual P. formosa and sexual Poecilia latipinna as a possible factor allowing for persistence and coexistence between the two sympatric species. Offspring of gynogenetic Amazon mollies did not differ significantly in survivorship compared to their sexual host, the Sailfin molly, P. latipinna. The presence of an adult female significantly reduced survival in both species, suggesting that filial cannibalisms operates in this system, but does not appear to play a role in stabilizing mixed sexual/asexual populations. Clark Hubbs, who spent 59 years at the University of Texas and was widely regarded as one of the state’s foremost researchers in the field of ichthyology, the study of fish, passed away February 3rd of 2008 after a long battle with colorectal cancer. He was 86.     

Comparative ecology of sexual and asexual gecko species (Lepidodactylus) in French Polynesia

Summary The asexual geckoLepidodactylus lugubris, its sexual congenerL. sp. (Takapoto) and hybrids between the two species inhabit the atoll of Takapoto, providing a natural experiment for studying co-existence and interactions between asexual and sexual populations. The range of the sexual species is confined to one section of the lagoon beach and the trees and buildings which abut it, whereas the asexual is distributed across the whole atoll and occupies many habitats. Behavioural experiments revealed no asymmetry in levels of aggression between the two species, suggesting that the confinement ofL. sp. (Takapoto) to the lagoon beach is not due to agonistic interactions. Ecological differences among the constituent clones of the asexual species exist but cannot completely account for the broader habitat use of the asexual. Within a single habitat, one clone ofL. lugubris consumes a wider range of prey items than its sexual relative. Other studies have found that the asexuals are extremely heterozygous relative to the sexuals; we hypothesize that their broad ecological tolerance may be attributable to heterosis. The co-existence of the sexual and parthenogenetic lizards on this small island seems to be stable and may be facilitated by the specialization of the sexual taxon to beach habitats.     

Diets of sexual and sperm‐dependent asexual dace (Chrosomus spp.): relevance to niche differentiation and mate choice hypotheses for coexistence

Sperm‐dependent asexual species must coexist with a sexual species (i.e. a sperm source) to reproduce. The maintenance of this coexistence, and hence the persistence of sperm‐dependent asexual species, may depend on ecological niche separation or preference by males for conspecific (i.e. sexual) mates. We first modified an analytical model to consider both of these mechanisms acting simultaneously on the coexistence of the two species. Our model indicates that a small amount of niche separation between parental species and hybrids can facilitate coexistence by weakening the requirement for male mate preference. We also estimated niche separation empirically in the Chrosomus (formerly Phoxinus) sexual‐asexual system based on diet overlap between sperm‐dependent asexuals and their two sexual host species. Diet overlap between the sexual species was not significant in either lake, whereas the sperm‐dependent asexual had an intermediate niche that overlapped significantly, but somewhat asymmetrically, with both sexual species. These empirical results were then used to parameterize our analytical model to predict the minimum strength of male mate preference required to maintain coexistence in each lake. Some male mate preference is likely required to maintain coexistence in the Chrosomus system, but the minimum required preference depends on the severity of density dependence. Future empirical work on understanding coexistence in sperm‐dependent asexual systems would benefit from taking both niche separation and mate choice into account, and from simultaneous empirical estimates of male mate choice, niche separation, and density dependence.     

Poor male function favours the coexistence of sexual and asexual relatives

Classical models of the evolution of sex typically assume that an asexual lineage, once derived, is reproductively separate from the sexual lineage from which it was derived. However, many asexuals, including hermaphrodite plants, produce male gametes capable of fertilising the eggs of co-existing sexuals, giving rise to sexual and asexual progeny. This male function of asexuals may be poor, and it has been proposed that this could favour sexuality and adversely affect the successful establishment of asexual lineages. We show that things are more complicated than this; the effect is frequency-dependent and poor male function may sometimes favour asexuality. In a spatially distributed population of flowering plants, it can prevent the successful invasion of either reproductive mode by the other via long-range dispersal. Consequently invasions must be driven by short-range dispersal, and are therefore extremely slow. Thus poor male function favours long-term co-existence of sexuals and asexuals. When coupled with the superior ability of asexuals to colonise virgin territory after an Ice Age, it may explain current ecological distribution patterns.     

Marginal distribution and high heterozygosity of asexual Caloglossa vieillardii (Delesseriaceae,Rhodophyta) along the Australian coasts

In animals and land plants, many asexual species originate through inter‐ or intraspecific crosses, and such heterozygous asexuals frequently are more abundant than their sexual relatives in marginal habitats. Although asexual species have been reported in various macroalgal taxa, detailed information regarding their distribution, heterozygosity, and origin is limited. Because many asexual tetrasporophyte strains of Caloglossa vieillardii have been isolated from South Australia, far from their core tropical habitats, we re‐examined the distribution range of asexual C. vieillardii and genotyped these and other western Pacific strains using an actin gene marker. We confirmed the marginal distribution of the asexuals; however, a small patch of sexual thalli was newly discovered 450 km further west from asexual populations in South Australia. Three heterozygous genotypes and one homozygous genotypes were detected from nine asexual populations; 21 heterozygous strains were obligately asexual, but one homozygous strain suddenly produced sexual gametophytes after several years of culture. We hypothesized that the most abundant heterozygous genotype (defined as type 3/4) in asexual populations occurred by a cross between type 3 and type 4 allele gametophytes, both of which were isolated from the Australian coasts. In the crossing experiments, certain combinations between type 3 females and type 4 males produced tetrasporophytes, which recycled successive tetrasporophytes. In the culture experiments, whereas both sexual and asexual strains successfully produced tetraspores at 12°C, no sexual strains released carpospores below 14°C. However, it is uncertain whether this slight difference of maturation temperature was related to the marginal distribution of asexual C. vieillardii.     

The potential role of chromosome telomere resetting consequent upon sex in the population dynamics of aphids: an hypothesis

Models of population structure have emphasized the importance of sex in maintaining lineages. This is because, despite the well known ‘two‐fold cost of sex’ compared with asex, it is considered that recombination rids the genome of accumulated mutations and increases its potential for adaptive variation. However, asexual lineages of eukaryotic organisms can also rapidly gain genetic variance directly by various mutational processes, thereby proving that so‐called ‘clones’ do not have strict genetic fidelity ( Lushai & Loxdale, 2002 ; Loxdale & Lushai, 2003a ), whereas the variation so produced may well have adaptive advantage during the evolutionary process. This being so, obligated asexuals or cyclical parthenogens that occasionally indulge in sexual recombination (‘rare sex’) cannot be deemed as ‘evolutionary dead‐ends’( Lushai, Loxdale & Allen, 2003a ). In addition, the persistence of asexual lineages (i.e. lineage longevity) may also involve the integrity of the telomere region, the physical end of the chromosomes ( Loxdale & Lushai, 2003b ). In this earlier study on this topic, we argued that the persistence and ultimate senescence of eukaryotic cell lineages (based upon the frequency of ‘capped’ and ‘uncapped’ chromosomes related to telomere functionality; Blackburn, 2000 ) may directly relate to the survival and persistence of lineages of whole asexual organisms. Aphids are a good model system to test this hypothesis because they show a variety of sexual/asexual reproductive strategies, whereas their mode of asexual reproduction is of the mitotic (= apomictic) type. We also suggested that many aphid lineages require occasional or even rare sexual recombination to re‐set telomere length to allow lineages to persist. Ample empirical evidence from diverse taxa, lineages, and different developmental stages now reveals that the telomere states are indeed re‐set by recombination (homologous or meiotic), thereby rejuvenating the lineage in question. The generational clock element of telomeric functionality has also been successfully described in artificially‐induced mammalian clonal systems. It thus appears that telomere function is a central molecular mechanism instigating and promoting lineage continuity per se. By contrast, we hypothesized that other long‐lived asexuals, or the rare category of ancient asexuals such as bdelloid rotifers, have compensatory mechanisms for maintaining chromosome functional integrity, which are somewhat different from conventional telomeric repeats. In the present study, we carry the analogy between eukaryotic cell functionality and aphid lineages a stage further. Here, we hypothesize that the changing frequency of capped and uncapped telomeres, progressing to senescence in a stochastic manner, may be an underlying factor that significantly contributes to population dynamics in asexual lineage evolution. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 90 , 719–728.     

Evolutionary history of contagious asexuality in Daphnia pulex

Asexual taxa are short-lived, suggesting that transitions to asexuality represent evolutionary dead-ends. However, with high rates of clonal origin and coexistence of asexuals and sexuals via selective asymmetries, asexuality may persist in the long term as a result of a dynamic equilibrium between clonal origin and extinction. Few such systems have been studied in detail. Here, we investigate the evolutionary history of asexual lineages of Daphnia pulex, which are derived from sexual relatives via the inheritance of a dominant female-limited meiosis-suppressing locus and inhabit ponds throughout northeastern North America (NA). Our extensive sampling and subsequent phylogenetic analysis using mitochondrial sequence data reveals a young and genetically diverse asexual assemblage, reflecting high rates of clonal origin due to the contagious nature of asexuality. Yet, asexuality is restricted to two phylogroups (B and C) with historical and/or present associations with northeastern NA and is absent from a northwestern phylogroup (A), supporting a recent northeastern origin of asexuality in this species. Furthermore, macrogeographic patterns of genetic variability indicate that phylogroups B and C recolonized northeastern NA from opposite directions, yet their presently overlapping geographic distributions are similarly divided into an eastern asexual and a western sexual region. We attribute these patterns to a recent contagious spread of asexuality from a northeastern source. If environment-mediated selective asymmetries play no significant role in determining the outcome of competitive interactions between sexuals and asexuals, regions of contact may be setting the stage for continued asexual conquests.     

Pollen profile,spatial structure,and access to sex in asexual hermaphrodites

Classical cost‐of‐sex models predict the rapid fixation of asexual reproduction. Coexistence of sexuals and asexuals is common among hermaphrodite plants, however, providing asexuals with access to sex via their male function; some of the sexually reproduced progeny they sire will be asexual. The ability of asexuals to sire progeny is often hindered by the production of poor quality pollen. Using cellular automata, it is shown that decreases in pollen quality in asexuals can greatly increase the period of coexistence of sexuals and asexuals and, consequently, the cumulative contribution of sex to asex. Extensive periods of coexistence are only likely, however, if pollen and seed are dispersed locally, in which case coexistence over thousands of generations can be achieved. It is argued that, with local dispersal, the negative relationship between pollen quality and the period of coexistence of sex and asex will result in patterns of geographic parthenogenesis in which asexuals that coexist with sexuals will exhibit a poor male function, whereas asexuals with a very efficient male function will occur in exclusively asexual populations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 954–966.     

Genetic tests of ancient asexuality in Root Knot Nematodes reveal recent hybrid origins

Background  

The existence of "ancient asexuals", taxa that have persisted for long periods of evolutionary history without sexual recombination, is both controversial and important for our understanding of the evolution and maintenance of sexual reproduction. A lack of sex has consequences not only for the ecology of the asexual organism but also for its genome. Several genetic signatures are predicted from long-term asexual (apomictic) reproduction including (i) large "allelic" sequence divergence (ii) lack of phylogenetic clustering of "alleles" within morphological species and (iii) decay and loss of genes specific to meiosis and sexual reproduction. These genetic signatures can be hard to assess since it is difficult to demonstrate the allelic nature of very divergent sequences, divergence levels may be complicated by processes such as inter-specific hybridization, and genes may have secondary roles unrelated to sexual reproduction. Apomictic species of Meloidogyne root knot nematodes have been suggested previously to be ancient asexuals. Their relatives reproduce by meiotic parthenogenesis or facultative sexuality, which in combination with the abundance of nematode genomic sequence data, makes them a powerful system in which to study the consequences of reproductive mode on genomic divergence.     

Relative contributions of sexual and asexual regeneration strategies in Populus nigra and Salix alba during the first years of establishment on a braided gravel bed river

Populus nigra L. var betulifolia and Salix alba L. var alba are early successional riparian tree species threatened throughout Continental Europe by significant changes to the natural physical processes governing their natural habitat – geomorphologically active floodplains. River management activities have dramatically altered natural patterns of river flow and rates of sediment delivery along rivers, with possible consequences for the balance between sexual and asexual regeneration strategies in these species. Conservation strategies will benefit from a greater understanding of the ways in which dynamic physical processes on the floodplain influence sexual and asexual recruitment. This paper describes a field survey investigating the relative abundance and spatial distributions of P. nigra and S. alba sexual and asexual recruits during the first years of establishment along a braided gravel bed river. Sexual and asexual recruits were identified by excavation along transects in a wet and a dry field season and distributional differences were described in terms of elevation on the floodplain, local sediment type and exposure to floodwaters. Regeneration was overwhelmingly from seed in the first 2–3 years following recruitment, but poor survival rates among sexual recruits saw a shift in the relative abundance of regeneration strategies over time. In relating hydrological data to recruitment, unseasonal flood disturbances had a negative effect on recruitment from seed and a positive effect on vegetative regeneration. Seedlings were associated with fine sediment deposits and were restricted primarily to low elevations on the flood plain, while asexual recruits had a wider spatial distribution. Certain microsite types were unique to either regeneration strategy.