POSITIVE SELECTION DRIVES FASTER‐Z EVOLUTION IN SILKMOTHS

Genes linked to X or Z chromosomes, which are hemizygous in the heterogametic sex, are predicted to evolve at different rates than those on autosomes. This “faster‐X effect” can arise either as a consequence of hemizygosity, which leads to more efficient selection for recessive beneficial mutations in the heterogametic sex, or as a consequence of reduced effective population size of the hemizygous chromosome, which leads to increased fixation of weakly deleterious mutations due to genetic drift. Empirical results to date suggest that, while the overall pattern across taxa is complicated, systems with male heterogamy show a faster‐X effect attributable to more efficient selection, whereas the faster‐Z effect in female‐heterogametic taxa is attributable to increased drift. To test the generality of the faster‐Z pattern seen in birds and snakes, we sequenced the genome of the lepidopteran silkmoth Bombyx huttoni. We show that silkmoths experience faster‐Z evolution, but unlike in birds and snakes, the faster‐Z effect appears to be attributable to more efficient positive selection. These results suggest that female heterogamy alone is unlikely to explain the reduced efficacy of selection on vertebrate Z chromosomes. It is likely that many factors, including differences in overall effective population size, influence Z chromosome evolution.     

A cryptic heterogametic transition revealed by sex-linked DNA markers in Palearctic green toads

In sharp contrast to birds and mammals, most cold‐blooded vertebrates have homomorphic (morphologically undifferentiated) sex chromosomes. This might result either from recurrent X‐Y recombination (occurring e.g. during occasional events of sex reversal) or from frequent turnovers (during which sex‐determining genes are overthrown by new autosomal mutations). Evidence for turnovers is indeed mounting in fish, but very few have so far been documented in amphibians, possibly because of practical difficulties in identifying sex chromosomes. Female heterogamety (ZW) has long been established in Bufo bufo, based on sex reversal and crossing experiments. Here, we investigate a sex‐linked marker identified from a laboratory cross between Palearctic green toads (Bufo viridis subgroup). The F₁ offspring produced by a female Bufo balearicus and a male Bufo siculus were phenotypically sexed, displaying an even sex ratio. A sex‐specific marker detected in highly reproducible AFLP genotypes was cloned. Sequencing revealed a noncoding, microsatellite‐containing fragment. Reamplification and genotyping of families of this and a reciprocal cross showed B. siculus to be male heterogametic (XY) and suggested the same system for B. balearicus. Our results thus reveal a cryptic heterogametic transition within bufonid frogs and help explain patterns of hybrid fitness within the B. viridis subgroup. Turnovers of genetic sex‐determination systems may be more frequent in amphibians than previously thought and thus contribute to the prevalence of homomorphic sex chromosomes in this group.     

Low rates of X‐Y recombination,not turnovers,account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup)

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X‐Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex‐linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X‐Y recombination in males. Phylogenetic analyses of sex‐linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X‐Y homomorphy and fine‐scale sequence similarity in these species do not stem from recent sex‐chromosome turnovers, but from occasional X‐Y recombination.     

SEX‐CHROMOSOME TURNOVERS INDUCED BY DELETERIOUS MUTATION LOAD

In sharp contrast with mammals and birds, many cold‐blooded vertebrates present homomorphic sex chromosomes. Empirical evidence supports a role for frequent turnovers, which replace nonrecombining sex chromosomes before they have time to decay. Three main mechanisms have been proposed for such turnovers, relying either on neutral processes, sex‐ratio selection, or intrinsic benefits of the new sex‐determining genes (due, e.g., to linkage with sexually antagonistic mutations). Here, we suggest an additional mechanism, arising from the load of deleterious mutations that accumulate on nonrecombining sex chromosomes. In the absence of dosage compensation, this load should progressively lower survival rate in the heterogametic sex. Turnovers should occur when this cost outweighs the benefits gained from any sexually antagonistic genes carried by the nonrecombining sex chromosome. We use individual‐based simulations of a Muller's ratchet process to test this prediction, and investigate how the relevant parameters (effective population size, strength and dominance of deleterious mutations, size of nonrecombining segment, and strength of sexually antagonistic selection) are expected to affect the rate of turnovers.     

Extreme heterochiasmy and nascent sex chromosomes in European tree frogs

We investigated sex-specific recombination rates in Hyla arborea, a species with nascent sex chromosomes and male heterogamety. Twenty microsatellites were clustered into six linkage groups, all showing suppressed or very low recombination in males. Seven markers were sex linked, none of them showing any sign of recombination in males (r=0.00 versus 0.43 on average in females). This opposes classical models of sex chromosome evolution, which envision an initially small differential segment that progressively expands as structural changes accumulate on the Y chromosome. For autosomes, maps were more than 14 times longer in females than in males, which seems the highest ratio documented so far in vertebrates. These results support the pleiotropic model of Haldane and Huxley, according to which recombination is reduced in the heterogametic sex by general modifiers that affect recombination on the whole genome.     

Mating ecology explains patterns of genome elimination

Genome elimination – whereby an individual discards chromosomes inherited from one parent, and transmits only those inherited from the other parent – is found across thousands of animal species. It is more common in association with inbreeding, under male heterogamety, in males, and in the form of paternal genome elimination. However, the reasons for this broad pattern remain unclear. We develop a mathematical model to determine how degree of inbreeding, sex determination, genomic location, pattern of gene expression and parental origin of the eliminated genome interact to determine the fate of genome‐elimination alleles. We find that: inbreeding promotes paternal genome elimination in the heterogametic sex; this may incur population extinction under female heterogamety, owing to eradication of males; and extinction is averted under male heterogamety, owing to countervailing sex‐ratio selection. Thus, we explain the observed pattern of genome elimination. Our results highlight the interaction between mating system, sex‐ratio selection and intragenomic conflict.     

THE VARIABILITY IS IN THE SEX CHROMOSOMES

Sex differences in the mean trait expression are well documented, not only for traits that are directly associated with reproduction. Less is known about how the variability of traits differs between males and females. In species with sex chromosomes and dosage compensation, the heterogametic sex is expected to show larger trait variability (“sex‐chromosome hypothesis”), yet this central prediction, based on fundamental genetic principles, has never been evaluated in detail. Here we show that in species with heterogametic males, male variability in body size is significantly larger than in females, whereas the opposite can be shown for species with heterogametic females. These results support the prediction of the sex‐chromosome hypothesis that individuals of the heterogametic sex should be more variable. We argue that the pattern demonstrated here for sex‐specific body size variability is likely to apply to any trait and needs to be considered when testing predictions about sex‐specific variability and sexual selection.     

Invasion and fixation of sex-reversal genes

We simulated a meta-population with random dispersal among demes but local mating within demes to investigate conditions under which a dominant female-determining gene W, with no individual selection advantage, can invade and become fixed in females, changing the population from male to female heterogamety. Starting with one mutant W in a single deme, the interaction of sex ratio selection and random genetic drift causes W to be fixed among females more often than a comparable neutral mutation with no influence on sex determination, even when YY males have slightly reduced viability. Meta-population structure and interdeme selection can also favour the fixation of W. The reverse transition from female to male heterogamety can also occur with higher probability than for a comparable neutral mutation. These results help to explain the involvement of sex-determining genes in the evolution of sex chromosomes and in sexual selection and speciation.     

Meiotic pairing constraints and the activity of sex chromosomes

The state of activity and condensation of the sex chromosomes in gametocytes is frequently different from that found in somatic cells. For example, whereas the X chromosomes of XY males are euchromatic and active in somatic cells, they are usually condensed and inactive at the onset of meiosis; in the somatic cells of female mammals, one X chromosome is heterochromatic and inactive, but both X chromosomes are euchromatic and active early in meiosis. In species in which the female is the heterogametic sex (ZZ males and ZW females), the W chromosome, which is often seen as a condensed chromatin body in somatic cells, becomes euchromatic in early oocytes. We describe an hypothesis which can explain these changes in the activity and condensation of sex chromosomes in gametocytes. It is based on the fact that normal chromosome pairing seems to be essential for the survival of sex cells; chromosomal anomalies resulting in incomplete pairing during meiosis usually result in gametogenic loss. We argue that the changes seen in the sex chromosomes reflect the need to avoid pairing failure during meiosis. Pairing normally requires structural and conformational homology of the two chromosomes, but when the regions is avoided when these regions become heterochromatinized. This hypothesis provides an explanation for the changes found in gametocytes both in species with male heterogamety and those with female heterogamety. It also suggests possible reasons for the frequent origin of large supernumerary chromosomes from sex chromosomes, and for the reported lack of dosage compensation in species with female heterogamety.     

ZW,XY, and yet ZW: Sex chromosome evolution in snakes even more complicated

Snakes are historically important in the formulation of several central concepts on the evolution of sex chromosomes. For over 50 years, it was believed that all snakes shared the same ZZ/ZW sex chromosomes, which are homomorphic and poorly differentiated in “basal” snakes such as pythons and boas, while heteromorphic and well differentiated in “advanced” (caenophidian) snakes. Recent molecular studies revealed that differentiated sex chromosomes are indeed shared among all families of caenophidian snakes, but that boas and pythons evolved likely independently male heterogamety (XX/XY sex chromosomes). The historical report of heteromorphic ZZ/ZW sex chromosomes in a boid snake was previously regarded as ambiguous. In the current study, we document heteromorphic ZZ/ZW sex chromosomes in a boid snake. A comparative approach suggests that these heteromorphic sex chromosomes evolved very recently and that they are poorly differentiated at the sequence level. Interestingly, two snake lineages with confirmed male heterogamety possess homomorphic sex chromosomes, but heteromorphic sex chromosomes are present in both snake lineages with female heterogamety. We point out that this phenomenon is more common across squamates. The presence of female heterogamety in non‐caenophidian snakes indicates that the evolution of sex chromosomes in this lineage is much more complex than previously thought, making snakes an even better model system for the evolution of sex chromosomes.     

Sex ratio selection and multi-factorial sex determination in the housefly: a dynamic model

Sex determining (SD) mechanisms are highly variable between different taxonomic groups and appear to change relatively quickly during evolution. Sex ratio selection could be a dominant force causing such changes. We investigate theoretically the effect of sex ratio selection on the dynamics of a multi-factorial SD system. The system considered resembles the naturally occurring three-locus system of the housefly, which allows for male heterogamety, female heterogamety and a variety of other mechanisms. Sex ratio selection is modelled by assuming cost differences in the production of sons and daughters, a scenario leading to a strong sex ratio bias in the absence of constraints imposed by the mechanism of sex determination. We show that, despite of the presumed flexibility of the SD system considered, equilibrium sex ratios never deviate strongly from 1 : 1. Even if daughters are very costly, a male-biased sex ratio can never evolve. If sons are more costly, sex ratio can be slightly female biased but even in case of large cost differences the bias is very small (<10% from 1 : 1). Sex ratio selection can lead to a shift in the SD mechanism, but cannot be the sole cause of complete switches from one SD system to another. In fact, more than one locus remains polymorphic at equilibrium. We discuss our results in the context of evolution of the variable SD mechanism found in natural housefly populations.     

Sex chromosome inversions enforce reproductive isolation across an avian hybrid zone

Across hybrid zones, the sex chromosomes are often more strongly differentiated than the autosomes. This is regularly attributed to the greater frequency of reproductive incompatibilities accumulating on sex chromosomes and their exposure in the heterogametic sex. Working within an avian hybrid zone, we explore the possibility that chromosome inversions differentially accumulate on the Z chromosome compared to the autosomes and thereby contribute to Z chromosome differentiation. We analyse the northern Australian hybrid zone between two subspecies of the long‐tailed finch (Poephila acuticauda), first described based on differences in bill colour, using reduced‐representation genomic sequencing for 293 individuals over a 1,530‐km transect. Autosomal differentiation between subspecies is minimal. In contrast, 75% of the Z chromosome is highly differentiated and shows a steep genomic cline, which is displaced 350 km to the west of the cline in bill colour. Differentiation is associated with two or more putative chromosomal inversions, each predominating in one subspecies. If inversions reduce recombination between hybrid incompatibilities, they are selectively favoured and should therefore accumulate in hybrid zones. We argue that this predisposes inversions to differentially accumulate on the Z chromosome. One genomic region affecting bill colour is on the Z, but the main candidates are on chromosome 8. This and the displacement of the bill colour and Z chromosome cline centres suggest that bill colour has not strongly contributed to inversion accumulation. Based on cline width, however, the Z chromosome and bill colour both contribute to reproductive isolation established between this pair of subspecies.     

Genetic conflict and changes in heterogametic mechanisms of sex determination

The consequences of cytoplasmic sex‐ratio distortion and host repression for the evolution of host sex‐determining mechanisms are examined. Analytical models and simulations are developed to investigate whether the interplay between sex‐ratio distorters and host masculinizers or resistance genes can cause heterogamety switching (changes between male and female heterogamety). Switches from female heterogamety to a system analogous to male heterogamety can occur when selection favours the spread of autosomal masculinizers. However, the evolutionary outcome depends on the type of repressor and costs associated with repression, and also on aspects of population structure. Under most conditions, systems evolved to a polymorphic sex‐determining state although many systems were characterized by numerical dominance of male heterogamety.     

THE EVOLUTION OF HETEROMORPHIC SEX CHROMOSOMES

The facts and ideas which have been discussed lead to the following synthesis and model. 1. Heteromorphic sex chromosomes evolved from a pair of homomorphic chromosomes which had an allelic difference at the sex-determining locus. 2. The first step in the evolution of sex-chromosome heteromorphism involved either a conformational or a structural difference between the homologues. A structural difference could have arisen through a rearrangement such as an inversion or a translocation. A conformational difference could have occurred if the sex-determining locus was located in a chromosomal domain which behaved as a single control unit and involved a substantial segment of the chromosome. It is assumed that any conformational difference present in somatic cells would have been maintained in meiotic prophase. 3. Lack of conformational or structural homology between the sex chromosomes led to meiotic pairing failure. Since pairing failure reduced fertility, mechanisms preventing it had a selective advantage. Meiotic inactivation (heterochromatinization) of the differential region of the X chromosome in species with heterogametic males and euchromatinization of the W in species with heterogametic females are such mechanisms, and through them the pairing problems are avoided. 4. Structural and conformational differences between the sex chromosomes in the heterogametic sex reduced recombination. In heterogametic males recombination was reduced still further by the heterochromatinization of the X chromosome, which evolved in response to selection against meiotic pairing failure. 5. Suppression of recombination resulted in an increase in the mutation rate and an increased rate of fixation of deleterious mutations in the recombination-free chromosome regions. Functional degeneration of the genetically isolated regions of the Y and W was the result. In XY males this often led to further meiotic inactivation of the differential region of the X chromosome, and in this way an evolutionary positive-feedback loop may have been established. 6. Structural degeneration (loss of material) followed functional degeneration of Y or W chromosomes either because the functionally degenerate genes had deleterious effects which made their loss a selective advantage, or because shorter chromosomes were selectively neutral and became fixed by chance. 7. The evolutionary routes to sex-chromosome heteromorphism in groups with female heterogamety are more limited than in those with male heterogamety. Oocytes are usually large and long-lived, and are likely to need the products of X- or Z-linked genes. Meiotic inactivation of these chromosomes is therefore unlikely. In the oocytes of ZW females, meiotic pairing failure is avoided through euchromatinization of the W rather than heterochromatinization of the Z chromosome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

When Sex Chromosomes Recombine Only in the Heterogametic Sex: Heterochiasmy and Heterogamety in Hyla Tree Frogs

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.     

Dynamic sex chromosomes in Old World chameleons (Squamata: Chamaeleonidae)

Much of our current state of knowledge concerning sex chromosome evolution is based on a handful of ‘exceptional’ taxa with heteromorphic sex chromosomes. However, classifying the sex chromosome systems of additional species lacking easily identifiable, heteromorphic sex chromosomes is indispensable if we wish to fully understand the genesis, degeneration and turnover of vertebrate sex chromosomes. Squamate reptiles (lizards and snakes) are a potential model clade for studying sex chromosome evolution as they exhibit a suite of sex‐determining modes yet most species lack heteromorphic sex chromosomes. Only three (of 203) chameleon species have identified sex chromosome systems (all with female heterogamety, ZZ/ZW). This study uses a recently developed method to identify sex‐specific genetic markers from restriction site‐associated DNA sequence (RADseq) data, which enables the identification of sex chromosome systems in species lacking heteromorphic sex chromosomes. We used RADseq and subsequent PCR validation to identify an XX/XY sex chromosome system in the veiled chameleon (Chamaeleo calyptratus), revealing a novel transition in sex chromosome systems within the Chamaeleonidae. The sex‐specific genetic markers identified here will be essential in research focused on sex‐specific, comparative, functional and developmental evolutionary questions, further promoting C. calyptratus’ utility as an emerging model organism.     

Sex differences in life span: Females homozygous for the X chromosome do not suffer the shorter life span predicted by the unguarded X hypothesis

Life span differs between the sexes in many species. Three hypotheses to explain this interesting pattern have been proposed, involving different drivers: sexual selection, asymmetrical inheritance of cytoplasmic genomes, and hemizygosity of the X(Z) chromosome (the unguarded X hypothesis). Of these, the unguarded X has received the least experimental attention. This hypothesis suggests that the heterogametic sex suffers a shortened life span because recessive deleterious alleles on its single X(Z) chromosome are expressed unconditionally. In Drosophila melanogaster, the X chromosome is unusually large (～20% of the genome), providing a powerful model for evaluating theories involving the X. Here, we test the unguarded X hypothesis by forcing D. melanogaster females from a laboratory population to express recessive X‐linked alleles to the same degree as males, using females exclusively made homozygous for the X chromosome. We find no evidence for reduced life span or egg‐to‐adult viability due to X homozygozity. In contrast, males and females homozygous for an autosome both suffer similar, significant reductions in those traits. The logic of the unguarded X hypothesis is indisputable, but our results suggest that the degree to which recessive deleterious X‐linked alleles depress performance in the heterogametic sex appears too small to explain general sex differences in life span.     

Novel evolutionary pathways of sex‐determining mechanisms

Evolutionary transitions between sex‐determining mechanisms (SDMs) are an enigma. Among vertebrates, individual sex (male or female) is primarily determined by either genes (genotypic sex determination, GSD) or embryonic incubation temperature (temperature‐dependent sex determination, TSD), and these mechanisms have undergone repeated evolutionary transitions. Despite this evolutionary lability, transitions from GSD (i.e. from male heterogamety, XX/XY, or female heterogamety, ZZ/ZW) to TSD are an evolutionary conundrum, as they appear to require crossing a fitness valley arising from the production of genotypes with reduced viability owing to being homogametic for degenerated sex chromosomes (YY or WW individuals). Moreover, it is unclear whether alternative (e.g. mixed) forms of sex determination can persist across evolutionary time. It has previously been suggested that transitions would be easy if temperature‐dependent sex reversal (e.g. XX male or XY female) was asymmetrical, occurring only in the homogametic sex. However, only recently has a mechanistic model of sex determination emerged that may allow such asymmetrical sex reversal. We demonstrate that selection for TSD in a realistic sex‐determining system can readily drive evolutionary transitions from GSD to TSD that do not require the production of YY or WW individuals. In XX/XY systems, sex reversal (female to male) occurs in a portion of the XX individuals only, leading to the loss of the Y allele (or chromosome) from the population as XX individuals mate with each other. The outcome is a population of XX individuals whose sex is determined by incubation temperature (TSD). Moreover, our model reveals a novel evolutionarily stable state representing a mixed‐mechanism system that has not been revealed by previous approaches. This study solves two long‐standing puzzles of the evolution of sex‐determining mechanisms by illuminating the evolutionary pathways and endpoints.     

The ecology and evolution of temperature‐dependent reaction norms for sex determination in reptiles: a mechanistic conceptual model

Sex‐determining mechanisms are broadly categorised as being based on either genetic or environmental factors. Vertebrate sex determination exhibits remarkable diversity but displays distinct phylogenetic patterns. While all eutherian mammals possess XY male heterogamety and female heterogamety (ZW) is ubiquitous in birds, poikilothermic vertebrates (fish, amphibians and reptiles) exhibit multiple genetic sex‐determination (GSD) systems as well as environmental sex determination (ESD). Temperature is the factor controlling ESD in reptiles and temperature‐dependent sex determination (TSD) in reptiles has become a focal point in the study of this phenomenon. Current patterns of climate change may cause detrimental skews in the population sex ratios of reptiles exhibiting TSD. Understanding the patterns of variation, both within and among populations and linking such patterns with the selection processes they are associated with, is the central challenge of research aimed at predicting the capacity of populations to adapt to novel conditions. Here we present a conceptual model that innovates by defining an individual reaction norm for sex determination as a range of incubation temperatures. By deconstructing individual reaction norms for TSD and revealing their underlying interacting elements, we offer a conceptual solution that explains how variation among individual reaction norms can be inferred from the pattern of population reaction norms. The model also links environmental variation with the different patterns of TSD and describes the processes from which they may arise. Specific climate scenarios are singled out as eco‐evolutionary traps that may lead to demographic extinction or a transition to either male or female heterogametic GSD. We describe how the conceptual principles can be applied to interpret TSD data and to explain the adaptive capacity of TSD to climate change as well as its limits and the potential applications for conservation and management programs.