Ovotestes suggest cryptic genetic influence in a reptile model for temperature-dependent sex determination

Sex determination and differentiation in reptiles is complex. Temperature-dependent sex determination (TSD), genetic sex determination (GSD) and the interaction of both environmental and genetic cues (sex reversal) can drive the development of sexual phenotypes. The jacky dragon (Amphibolurus muricatus) is an attractive model species for the study of gene–environment interactions because it displays a form of Type II TSD, where female-biased sex ratios are observed at extreme incubation temperatures and approximately 50 : 50 sex ratios occur at intermediate temperatures. This response to temperature has been proposed to occur due to underlying sex determining loci, the influence of which is overridden at extreme temperatures. Thus, sex reversal at extreme temperatures is predicted to produce the female-biased sex ratios observed in A. muricatus. The occurrence of ovotestes during development is a cellular marker of temperature sex reversal in a closely related species Pogona vitticeps. Here, we present the first developmental data for A. muricatus, and show that ovotestes occur at frequencies consistent with a mode of sex determination that is intermediate between GSD and TSD. This is the first evidence suggestive of underlying unidentified sex determining loci in a species that has long been used as a model for TSD.     

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.     

High elevation increases the risk of Y chromosome loss in Alpine skink populations with sex reversal

The view that has genotypic sex determination and environmental sex determination as mutually exclusive states in fishes and reptiles has been contradicted by the discovery that chromosomal sex and environmental influences can co-exist within the same species, hinting at a continuum of intermediate states. Systems where genes and the environment interact to determine sex present the opportunity for sex reversal to occur, where the phenotypic sex is the opposite of that predicted by their sex chromosome complement. The skink Bassiana duperreyi has XX/XY sex chromosomes with sex reversal of the XX genotype to a male phenotype, in laboratory experiments, and in field nests, in response to exposure to cold incubation temperatures. Here we studied the frequency of sex reversal in adult populations of B. duperreyi in response to climatic variation, using elevation as a surrogate for environmental temperatures. We demonstrate sex reversal in the wild for the first time in adults of a reptile species with XX/XY sex determination. The highest frequency of sex reversal occurred at the highest coolest elevation location, Mt Ginini (18.46%) and decreased in frequency to zero with decreasing elevation. We model the impact of this under Fisher’s frequency-dependent selection to show that, at the highest elevations, populations risk the loss of the Y chromosome and a transition to temperature-dependent sex determination. This study contributes to our understanding of the risks of extinction from climate change in species subject to sex reversal by temperature, and will provide focus for future research to test on-the-ground management strategies to mitigate the effects of climate in local populations.Subject terms: Evolutionary biology, Population genetics     

Mating strategies in two species of dart-poison frogs: a comparative study

Comparative field studies of species of dart-poison frogs in the genus Dendrobates were carried out to test predictions from two hypotheses that attempt to explain female-female competition for mates in species of Dendrobates with male parental care. The sex role reversal hypothesis proposes that males invest so much time and energy in parental care that receptive males are rare relative to receptive females, and females compete to find and mate with receptive males. The parental quality hypothesis proposes that females compete to monopolize the parental effort of particular males, because they potentially suffer a cost when their mates care for the offspring of other females. Comparisons between species with male parental care (Dendrobates leucomelas) and female parental care (Dendrobates histrionicus) contradicted prediction of the sex role reversal hypothesis, but were consistent with predictions of the parental quality hypothesis. Male D. histrionicus did not compete for mates more aggressively than male D. leucomelas, and male D. leucomelas were not more selective about mating than male D. histrionicus. Female D. leucomelas and D. histrionicus were both selective about mating; female D. leucomelas associated with and competed for particular males, whereas female D. histrionicus did not.     

Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development

It has been extensively documented that exposure of amphibians and teleost fish to exogenous steroid hormones like estrogen, androgen, xenoestrogen or steroid biosynthesis inhibitors can impair their gonadal development or induce sex reversal against genotypic sex. However, the molecular pathways underlying sexual development and the effects of sex steroids or other exogenous hormones in these aquatic vertebrates remain elusive. Recently, a germ plasm-associated piRNA (piwi-interacting RNA) pathway has been shown to be a determinant in the development of animal gonadal germline cells. In the current study, we examined whether this piRNA pathway is involved in the regulation of sex steroid hormones in gonadal development. We firstly established developmental expression patterns of three key piRNA pathway genes (mael, piwi and vasa), during Silurana (Xenopus) tropicalis embryogenesis and early larval development. All three genes exhibit high expression at early developmental stages and have significantly decreased expression thereafter, indicating a very active involvement of piRNA pathway at the beginning of embryogenesis. We further examined gene expression changes of those genes in frog larvae exposed to two sex steroid biosynthesis inhibitors, fadrozole and finasteride, both of which are known to result in male-biased or female-biased phenotypes, respectively. We found that fadrozole and finasteride exposures increased the expression of piRNA pathway genes such as mael and vasa at the larval stage when the expression of piRNA pathway genes is programmed to be very low. Therefore, our results indicate that the piRNA pathway is likely a common pathway by which different sex steroid hormones regulate gonadal sex differentiation.     

Inferring the Genetic Basis of Sex Determination from the Genome of a Dioecious Nightshade

Dissecting the genetic mechanisms underlying dioecy (i.e., separate female and male individuals) is critical for understanding the evolution of this pervasive reproductive strategy. Nonetheless, the genetic basis of sex determination remains unclear in many cases, especially in systems where dioecy has arisen recently. Within the economically important plant genus Solanum (∼2,000 species), dioecy is thought to have evolved independently at least 4 times across roughly 20 species. Here, we generate the first genome sequence of a dioecious Solanum and use it to ascertain the genetic basis of sex determination in this species. We de novo assembled and annotated the genome of Solanum appendiculatum (assembly size: ∼750 Mb scaffold N50: 0.92 Mb; ∼35,000 genes), identified sex-specific sequences and their locations in the genome, and inferred that males in this species are the heterogametic sex. We also analyzed gene expression patterns in floral tissues of males and females, finding approximately 100 genes that are differentially expressed between the sexes. These analyses, together with observed patterns of gene-family evolution specific to S. appendiculatum, consistently implicate a suite of genes from the regulatory network controlling pectin degradation and modification in the expression of sex. Furthermore, the genome of a species with a relatively young sex-determination system provides the foundational resources for future studies on the independent evolution of dioecy in this clade.     

Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans

X-linked sex-ratio distorters that disrupt spermatogenesis can cause a deficiency in functional Y-bearing sperm and a female-biased sex ratio. Y-linked modifiers that restore a normal sex ratio might be abundant and favored when a X-linked distorter is present. Here we investigated natural variation of Y-linked suppressors of sex-ratio in the Winters systems and the ability of these chromosomes to modulate gene expression in Drosophila simulans. Seventy-eight Y chromosomes of worldwide origin were assayed for their resistance to the X-linked sex-ratio distorter gene Dox. Y chromosome diversity caused males to sire ∼63% to ∼98% female progeny. Genome-wide gene expression analysis revealed hundreds of genes differentially expressed between isogenic males with sensitive (high sex ratio) and resistant (low sex ratio) Y chromosomes from the same population. Although the expression of about 75% of all testis-specific genes remained unchanged across Y chromosomes, a subset of post-meiotic genes was upregulated by resistant Y chromosomes. Conversely, a set of accessory gland-specific genes and mitochondrial genes were downregulated in males with resistant Y chromosomes. The D. simulans Y chromosome also modulated gene expression in XXY females in which the Y-linked protein-coding genes are not transcribed. The data suggest that the Y chromosome might exert its regulatory functions through epigenetic mechanisms that do not require the expression of protein-coding genes. The gene network that modulates sex ratio distortion by the Y chromosome is poorly understood, other than that it might include interactions with mitochondria and enriched for genes expressed in post-meiotic stages of spermatogenesis.     

Chromatin landscape associated with sexual differentiation in a UV sex determination system

In many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, sexual differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying the regulation of sex-biased expression of genes during sexual differentiation remain elusive. Here, we investigated the extent and nature of epigenomic changes associated with UV sexual differentiation in the brown alga Ectocarpus, a model UV system. Six histone modifications were quantified in near-isogenic lines, leading to the identification of 16 chromatin signatures across the genome. Chromatin signatures correlated with levels of gene expression and histone PTMs changes in males versus females occurred preferentially at genes involved in sex-specific pathways. Despite the absence of chromosome scale dosage compensation and the fact that UV sex chromosomes recombine across most of their length, the chromatin landscape of these chromosomes was remarkably different to that of autosomes. Hotspots of evolutionary young genes in the pseudoautosomal regions appear to drive the exceptional chromatin features of UV sex chromosomes.     

Sex-ratio adjustment in response to local mate competition is achieved through an alteration of egg size in a haplodiploid spider mite

Sex-ratio adjustments are commonly observed in haplodiploid species. However, the underlying proximate mechanisms remain elusive. We investigated these mechanisms in Tetranychus urticae, a haplodiploid spider mite known to adjust sex ratio in response to the level of local mate competition (LMC). In this species, egg size determines fertilization probability, with larger eggs being more likely to be fertilized, and thus become female. We explored the hypothesis that sex-ratio adjustment is achieved through adjustment of egg size. By using spider mites from a large population, we found that females produced not only a higher proportion of daughters under high levels of LMC, but also larger eggs. Moreover, in populations experimentally evolving under varying levels of LMC, both the proportion of females and the egg size increased with LMC intensity. These results suggest that sex-ratio adjustment in spider mites is mediated by egg size, although the causal relationship remains to be tested.     

Genetic Basis of a Violation of Dollo’s Law: Re-Evolution of Rotating Sex Combs in Drosophila bipectinata

Phylogenetic analyses suggest that violations of “Dollo’s law”—that is, re-evolution of lost complex structures—do occur, albeit infrequently. However, the genetic basis of such reversals has not been examined. Here, we address this question using the Drosophila sex comb, a recently evolved, male-specific morphological structure composed of modified bristles. In some species, sex comb development involves only the modification of individual bristles, while other species have more complex “rotated” sex combs that are shaped by coordinated migration of epithelial tissues. Rotated sex combs were lost in the ananassae species subgroup and subsequently re-evolved, ∼12 million years later, in Drosophila bipectinata and its sibling species. We examine the genetic basis of the differences in sex comb morphology between D. bipectinata and D. malerkotliana, a closely related species with a much simpler sex comb representing the ancestral condition. QTL mapping reveals that >50% of this difference is controlled by one chromosomal inversion that covers ∼5% of the genome. Several other, larger inversions do not contribute appreciably to the phenotype. This genetic architecture suggests that rotating sex combs may have re-evolved through changes in relatively few genes. We discuss potential developmental mechanisms that may allow lost complex structures to be regained.     

The autoregulatory loop: A common mechanism of regulation of key sex determining genes in insects

Sex determination in most insects is structured as a gene cascade, wherein a primary signal is passed through a series of sex-determining genes, culminating in a downstream double-switch known as doublesex that decides the sexual fate of the embryo. From the literature available on sex determination cascades, it becomes apparent that sex determination mechanisms have evolved rapidly. The primary signal that provides the cue to determine the sex of the embryo varies remarkably, not only among taxa, but also within taxa. Furthermore, the upstream key gene in the cascade also varies between species and even among closely related species. The order Insecta alone provides examples of astoundingly complex diversity of upstream key genes in sex determination mechanisms. Besides, unlike key upstream genes, the downstream double-switch gene is alternatively spliced to form functional sex-specific isoforms. This sex-specific splicing is conserved across insect taxa. The genes involved in the sex determination cascade such as Sex-lethal (Sxl) in Drosophila melanogaster, transformer (tra) in many other dipterans, coleopterans and hymenopterans, Feminizer (fem) in Apis mellifera, and IGF-II mRNA-binding protein (Bmimp) in Bombyx mori are reported to be regulated by an autoregulatory positive feedback loop. In this review, by taking examples from various insects, we propose the hypothesis that autoregulatory loop mechanisms of sex determination might be a general strategy. We also discuss the possible reasons for the evolution of autoregulatory loops in sex determination cascades and their impact on binary developmental choices.     

Physiological basis of interactive responses to temperature and salinity in coastal marine invertebrate: Implications for responses to warming

Developing physiological mechanistic models to predict species’ responses to climate‐driven environmental variables remains a key endeavor in ecology. Such approaches are challenging, because they require linking physiological processes with fitness and contraction or expansion in species’ distributions. We explore those links for coastal marine species, occurring in regions of freshwater influence (ROFIs) and exposed to changes in temperature and salinity. First, we evaluated the effect of temperature on hemolymph osmolality and on the expression of genes relevant for osmoregulation in larvae of the shore crab Carcinus maenas. We then discuss and develop a hypothetical model linking osmoregulation, fitness, and species expansion/contraction toward or away from ROFIs. In C. maenas, high temperature led to a threefold increase in the capacity to osmoregulate in the first and last larval stages (i.e., those more likely to experience low salinities). This result matched the known pattern of survival for larval stages where the negative effect of low salinity on survival is mitigated at high temperatures (abbreviated as TMLS). Because gene expression levels did not change at low salinity nor at high temperatures, we hypothesize that the increase in osmoregulatory capacity (OC) at high temperature should involve post‐translational processes. Further analysis of data suggested that TMLS occurs in C. maenas larvae due to the combination of increased osmoregulation (a physiological mechanism) and a reduced developmental period (a phenological mechanisms) when exposed to high temperatures. Based on information from the literature, we propose a model for C. maenas and other coastal species showing the contribution of osmoregulation and phenological mechanisms toward changes in range distribution under coastal warming. In species where the OC increases with temperature (e.g., C. maenas larvae), osmoregulation should contribute toward expansion if temperature increases; by contrast in those species where osmoregulation is weaker at high temperature, the contribution should be toward range contraction.