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.     

Sex chromosome turnovers and genetic drift: a simulation study

The recent advances of new genomic technologies have enabled the identification and characterization of sex chromosomes in an increasing number of nonmodel species, revealing that many plants and animals undergo frequent sex chromosome turnovers. What evolutionary forces drive these turnovers remains poorly understood, but it was recently proposed that drift might play a more important role than generally assumed. We analysed the dynamics of different types of turnovers using individual‐based simulations and show that when mediated by genetic drift, turnovers are usually easier to achieve than substitutions at neutral markers, but that their dynamics and relative likelihoods vary with the type of the resident and emergent sex chromosome system (XY and/or ZW) and the dominance relationships among the sex‐determining factors. Focusing on turnovers driven by epistatically dominant mutations, we find that drift‐mediated turnovers that preserve the heterogamety pattern are 2–4× more likely than those along which the heterogametic sex changes. This ratio nevertheless decreases along with effective population size and can even reverse in case of extreme polygyny. This can be attributed to a ‘drift‐induced’ selective force, known to influence transitions between male and female heterogamety, but which according to our study does not affect turnovers that preserve the heterogametic sex.     

The red bayberry genome and genetic basis of sex determination

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high‐quality genome for both a female and a male individual of red bayberry. The genome size was 313‐Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole‐genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59‐Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59‐Kb female‐specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non‐recombinant in the female‐specific region. Sex‐specific molecular markers developed from candidate genes co‐segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family.     

Identification of sex‐specific molecular markers using restriction site‐associated DNA sequencing

A major barrier to evolutionary studies of sex determination and sex chromosomes has been a lack of information on the types of sex‐determining mechanisms that occur among different species. This is particularly problematic in groups where most species lack visually heteromorphic sex chromosomes, such as fish, amphibians and reptiles, because cytogenetic analyses will fail to identify the sex chromosomes in these species. We describe the use of restriction site‐associated DNA (RAD) sequencing, or RAD‐seq, to identify sex‐specific molecular markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the lizard Anolis carolinensis. We performed RAD‐seq on seven male and ten female A. carolinensis and found one male‐specific molecular marker. Anolis carolinensis has previously been shown to possess male heterogamety and the recently published A. carolinensis genome facilitated the characterization of the sex‐specific RAD‐seq marker. We validated the male specificity of the new marker using PCR on additional individuals and also found that it is conserved in some other Anolis species. We discuss the utility of using RAD‐seq to identify sex‐determining mechanisms in other species with cryptic or homomorphic sex chromosomes and the implications for the evolution of male heterogamety in Anolis.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

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.     

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.     

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.     

Sex from W to Z: evolution of vertebrate sex chromosomes and sex determining genes

Sex determination in major vertebrate groups appears to be very variable, including systems of male heterogamety, female heterogamety and a variety of genetic and environmental sex determining systems. Yet comparative studies of sex chromosomes and sex determining genes now suggest that these differences are more apparent than real. The sex chromosomes of even widely divergent groups now appear to have changed very little over the last 300+ million years, and even independently derived sex chromosomes seem to have followed the same set of evolutionary rules. The sex determining pathway seems to be extremely conserved, although the control of the genes in this pathway is vested in different elements. We present a scenario for the independent evolution of XY male heterogamety in mammals and ZW female heterogamety in birds and some reptiles. We suggest that sex determining genes can be made redundant, and replaced by control at another step of a conserved sex determining pathway, and how choice of a gene as a sex switch has led to the evolution of new sex chromosome systems. J. Exp. Zool. 290:449-462, 2001.     

Heritable variation of sex ratio in a polychaete worm

Ophryotrocha labronica is a gonochoristic polychaete worm whose sex determining mechanism and sex ratio control are supposed to be polygenic. From a lab population, whose sex ratio (i.e., proportion of males) was 0.5, the estimate of sex ratio heritability by offspring-father regression was 0.54 ± 0.15 and by offspring-mother regression was not significantly different from 0. Estimate of sex ratio repeatability between successive broods of a pair was 0.64 ± 0.33. Since female parents do not contribute in any way to the variability of sex ratio, sex ratio variation seems to be largely a paternal character. On the basis of these estimates we advance the hypothesis that in this species sex is determined by a multilocus genetic system, allowing the combined effects of a female major sex gene (which could give rise to a form of female heterogamety) and masculinizing modifiers. The hypothesis that the male sex has the least canalised sexual differentiation is supported by the observation that some old males developed oocytes.     

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.     

How to identify sex chromosomes and their turnover

Although sex is a fundamental component of eukaryotic reproduction, the genetic systems that control sex determination are highly variable. In many organisms the presence of sex chromosomes is associated with female or male development. Although certain groups possess stable and conserved sex chromosomes, others exhibit rapid sex chromosome evolution, including transitions between male and female heterogamety, and turnover in the chromosome pair recruited to determine sex. These turnover events have important consequences for multiple facets of evolution, as sex chromosomes are predicted to play a central role in adaptation, sexual dimorphism, and speciation. However, our understanding of the processes driving the formation and turnover of sex chromosome systems is limited, in part because we lack a complete understanding of interspecific variation in the mechanisms by which sex is determined. New bioinformatic methods are making it possible to identify and characterize sex chromosomes in a diverse array of non‐model species, rapidly filling in the numerous gaps in our knowledge of sex chromosome systems across the tree of life. In turn, this growing data set is facilitating and fueling efforts to address many of the unanswered questions in sex chromosome evolution. Here, we synthesize the available bioinformatic approaches to produce a guide for characterizing sex chromosome system and identity simultaneously across clades of organisms. Furthermore, we survey our current understanding of the processes driving sex chromosome turnover, and highlight important avenues for future research.     

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.     

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.     

Hermaphroditism and gonochorism. a new hypothesis on the evolution of sexuality in crustacea

Most crustaceans are gonochoristic but hermaphroditism occurs in primitive classes as well as in different orders of higher Crustacea. Though studies have been carried out in plants and animals on the advantages of these two types of sexuality, it is not known how hermaphroditism can change into gonochorism and vice versa. The new hypothesis we report here is based on recent results on biased sex ratio in Crustacea. We suggest that ancestral sexuality was a simultaneous hermaphroditism as it exists still today in primitive groups. Gonochorism may have appeared following integration in the host genome of a parasitic xenogenous DNA inhibiting expression of ‘male genes’. Female sex would be anterior to male sex, and male heterogamety can be seen as a by-product of an intragenomic conflict in a species with an ancestral female heterogamety. Sequential hermaphroditism in higher Crustacea would be a secondary hermaphroditism resulting from other genetic conflicts between host genes and repressing heterochromosomic genes (parasitic DNA from xenogenous origin?)     

REPLICATED ORIGIN OF FEMALE‐BIASED ADULT SEX RATIO IN INTRODUCED POPULATIONS OF THE TRINIDADIAN GUPPY (POECILIA RETICULATA)

There are many theoretical and empirical studies explaining variation in offspring sex ratio but relatively few that explain variation in adult sex ratio. Adult sex ratios are important because biased sex ratios can be a driver of sexual selection and will reduce effective population size, affecting population persistence and shapes how populations respond to natural selection. Previous work on guppies (Poecilia reticulata) gives mixed results, usually showing a female‐biased adult sex ratio. However, a detailed analysis showed that this bias varied dramatically throughout a year and with no consistent sex bias. We used a mark‐recapture approach to examine the origin and consistency of female‐biased sex ratio in four replicated introductions. We show that female‐biased sex ratio arises predictably and is a consequence of higher male mortality and longer female life spans with little effect of offspring sex ratio. Inconsistencies with previous studies are likely due to sampling methods and sampling design, which should be less of an issue with mark‐recapture techniques. Together with other long‐term mark‐recapture studies, our study suggests that bias in offspring sex ratio rarely contributes to adult sex ratio in vertebrates. Rather, sex differences in adult survival rates and longevity determine vertebrate adult sex ratio.     

Evolutionary transitions between mechanisms of sex determination in vertebrates

Sex in many organisms is a dichotomous phenotype--individuals are either male or female. The molecular pathways underlying sex determination are governed by the genetic contribution of parents to the zygote, the environment in which the zygote develops or interaction of the two, depending on the species. Systems in which multiple interacting influences or a continuously varying influence (such as temperature) determines a dichotomous outcome have at least one threshold. We show that when sex is viewed as a threshold trait, evolution in that threshold can permit novel transitions between genotypic and temperature-dependent sex determination (TSD) and remarkably, between male (XX/XY) and female (ZZ/ZW) heterogamety. Transitions are possible without substantive genotypic innovation of novel sex-determining mutations or transpositions, so that the master sex gene and sex chromosome pair can be retained in ZW-XY transitions. We also show that evolution in the threshold can explain all observed patterns in vertebrate TSD, when coupled with evolution in embryonic survivorship limits.     

Within‐population polymorphism of sex‐determination systems in the common frog (Rana temporaria)

In sharp contrast with birds and mammals, the sex chromosomes of ectothermic vertebrates are often undifferentiated, for reasons that remain debated. A linkage map was recently published for Rana temporaria (Linnaeus, 1758) from Fennoscandia (Eastern European lineage), with a proposed sex‐determining role for linkage group 2 (LG2). We analysed linkage patterns in lowland and highland populations from Switzerland (Western European lineage), with special focus on LG2. Sibship analyses showed large differences from the Fennoscandian map in terms of recombination rates and loci order, pointing to large‐scale inversions or translocations. All linkage groups displayed extreme heterochiasmy (total map length was 12.2 cM in males, versus 869.8 cM in females). Sex determination was polymorphic within populations: a majority of families (with equal sex ratios) showed a strong correlation between offspring phenotypic sex and LG2 paternal haplotypes, whereas other families (some of which with female‐biased sex ratios) did not show any correlation. The factors determining sex in the latter could not be identified. This coexistence of several sex‐determination systems should induce frequent recombination of X and Y haplotypes, even in the absence of male recombination. Accordingly, we found no sex differences in allelic frequencies on LG2 markers among wild‐caught male and female adults, except in one high‐altitude population, where nonrecombinant Y haplotypes suggest sex to be entirely determined by LG2. Multifactorial sex determination certainly contributes to the lack of sex‐chromosome differentiation in amphibians.     

Intragenomic conflict produces sex ratio dynamics that favor maternal sex ratio distorters

Maternal sex ratio distorters (MSDs) are selfish elements that enhance their transmission by biasing their host's sex allocation in favor of females. While previous models have predicted that the female‐biased populations resulting from sex ratio distortion can benefit from enhanced productivity, these models neglect Fisherian selection for nuclear suppressors, an unrealistic assumption in most systems. We used individual‐based computer simulation modeling to explore the intragenomic conflict between sex ratio distorters and their suppressors and explored the impacts of these dynamics on population‐level competition between species characterized by MSDs and those lacking them. The conflict between distorters and suppressors was capable of producing large cyclical fluctuations in the population sex ratio and reproductive rate. Despite fitness costs associated with the distorters and suppressors, MSD populations often exhibited enhanced productivity and outcompeted non‐MSD populations in single and multiple‐population competition simulations. Notably, the conflict itself is beneficial to the success of populations, as sex ratio oscillations limit the competitive deficits associated with prolonged periods of male rarity. Although intragenomic conflict has been historically viewed as deleterious to populations, our results suggest that distorter–suppressor conflict can provide population‐level advantages, potentially helping to explain the persistence of sex ratio distorters in a range of taxa.     

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.