Sex has profoundly shaped animal biology and evolution, yet sex determination is strikingly diverse and its evolutionary origins remain poorly understood outside bilaterians. We investigate the molecular basis of sex determination in eight gonochoristic sponge species using integrated genomic and transcriptomic analyses. We identify sex chromosomes in two species, likely arising through chromosomal rearrangements, and find widespread polygenic sex determination across all taxa. Sponge sex determination relies on a deeply conserved genetic toolkit with sex-specific loci in DNA repair and recombination genes, syntenic across species and traceable to unicellular ancestors, indicating that core components of sexual reproduction predate animals. We also uncover a younger metazoan-specific complement and show independent evolution of XY and ZW systems, revealing recurrent shifts in heterogamety.

Sex-linked meiotic drivers limit the inheritance of the alternate sex chromosome in the heterogametic sex, subsequently skewing the offspring sex ratio. They consequently have large impacts on genome evolution, adaptation, and the emergence and maintenance of sexually selected traits. Despite this, our understanding of their molecular basis and consequences for gametogenesis and sex chromosome regulation more broadly has focused on a handful of model organisms, primarily Drosophila and mouse, which are not representative of the broad diversity of reproductive modes and drive systems in nature. Here, we employ single-cell RNA sequencing (scRNA-seq) to investigate a sex-linked meiotic driver in the Malaysian stalk-eyed fly, Teleopsis dalmanni. First, we produce a comprehensive single-cell atlas of the male T. dalmanni gonad and identify major testis cell types. We then provide a comprehensive profile of the cellular and transcriptional landscape of the testis, providing evidence for a lack of complete meiotic sex chromosome inactivation and complex trajectory of dosage compensation. Second, by contrasting single-cell expression data between drive and standard testes, we provide insight into the consequences of a meiotic driver for the transcriptomic landscape of the testis and sex chromosome regulation. Importantly, we show that the presence of a meiotic driver does not perturb fundamental patterns of X-linked regulation. Our results provide insight into how the meiotic driver might bias its transmission to the next generation and highlight genes with perturbed expression as a potential consequence of the disruption of spermatogenesis.

Sex chromosomes follow distinct evolutionary trajectories compared to the rest of the genome. In many cases, sex chromosomes (X and Y or Z and W) significantly differentiate from one another resulting in heteromorphic sex chromosome systems. Such heteromorphic systems are thought to act as an evolutionary trap that prevents subsequent turnover of the sex chromosome system. For old, degenerated sex chromosome systems, chromosomal fusion with an autosome may be one way that sex chromosomes can "refresh" their sequence content. We investigated these dynamics using treehoppers (hemipteran insects of the family Membracidae), which ancestrally have XX/X0 sex chromosomes. We assembled the most complete reference assembly for treehoppers to date for Umbonia crassicornis and employed comparative genomic analyses of 12 additional treehopper species to analyze X chromosome variation across different evolutionary timescales. We find that the X chromosome is largely conserved, with one exception being an X-autosome fusion in Calloconophora caliginosa. We also compare the ancestral treehopper X with other X chromosomes in Auchenorrhyncha (the clade containing treehoppers, leafhoppers, spittlebugs, cicadas, and planthoppers), revealing X conservation across more than 300 million years. These findings shed light on chromosomal evolution dynamics in treehoppers and the role of chromosomal rearrangements in sex chromosome evolution.

Genomic conflict between the sexes over shared traits is widely assumed to be resolved through the evolution of sex-biased expression and the subsequent emergence of sexually dimorphic phenotypes. However, while there is support for a broad relationship between genome-wide patterns of expression level and sexual conflict, recent studies suggest that sex differences in the nature and strength of interactions between loci are instead key to conflict resolution. Furthermore, the advent of new technologies for measuring and perturbing expression means we now have much more power to detect genomic signatures of sexual conflict. Here, we review our current understanding of the genomic architecture of sexual conflict in the light of these new studies and highlight the potential for novel approaches to address outstanding knowledge gaps.

Studies of sex chromosome systems at early stages of divergence are key to understanding the initial process and underlying causes of recombination suppression. However, identifying signatures of divergence in homomorphic sex chromosomes can be challenging due to high levels of sequence similarity between the X and the Y. Variations in methodological precision and underlying data can make all the difference between detecting subtle divergence patterns or missing them entirely. Recent efforts to test for X-Y sequence differentiation in the guppy have led to contradictory results. Here, we apply different analytical methodologies to the same data set to test for the accuracy of different approaches in identifying patterns of sex chromosome divergence in the guppy. Our comparative analysis reveals that the most substantial source of variation in the results of the different analyses lies in the reference genome used. Analyses using custom-made genome assemblies for the focal population or species successfully recover a signal of divergence across different methodological approaches. By contrast, using the distantly related Xiphophorus reference genome results in variable patterns, due to both sequence evolution and structural variations on the sex chromosomes between the guppy and Xiphophorus. Changes in mapping and filtering parameters can additionally introduce noise and obscure the signal. Our results illustrate how analytical differences can alter perceived results and we highlight best practices for the study of nascent sex chromosomes.

Sex chromosomes have evolved frequently across the tree of life, and have been a source of fascination for decades due to their unique evolutionary trajectories. They are hypothesised to be important drivers in a broad spectrum of biological processes and are the focus of a rich body of evolutionary theory. Whole-genome sequencing provides exciting opportunities to test these theories through contrasts between independently evolved sex chromosomes across the full spectrum of their evolutionary lifecycles. However, identifying sex chromosomes, particularly nascent ones, is challenging, often requiring specific combinations of methodologies. This is a major barrier to progress in the field and can result in discrepancies between studies that apply different approaches. Currently, no single pipeline exists to integrate data across these methods in a statistical framework to identify sex chromosomes at all ages and levels of sequence divergence. To address this, we present SexFindR, a comprehensive workflow to improve robustness and transparency in identifying sex-linked sequences. We validate our approach using publicly available data from five species that span the continuum of sex chromosome divergence, from homomorphic sex chromosomes with only a single SNP that determines sex, to heteromorphic sex chromosomes with extensive degeneration. Next, we apply SexFindR to our large-scale population genomics dataset for sea lamprey, a jawless vertebrate whose sex determination system remains a mystery despite decades of research. We decisively show that sea lamprey do not harbour sex-linked sequences in their somatic genome, leaving open the possibility that sex is determined environmentally or within the germline genome. ### Competing Interest Statement The authors have declared no competing interest.

Differences in allele frequencies at autosomal genes between males and females in a population can result from two scenarios. First, unresolved sexual conflict over survival can produce allelic differentiation between the sexes. However, given the substantial mortality costs required to produce allelic differences between males and females at each generation, it remains unclear how many loci within the genome experience significant sexual conflict over survival. Alternatively, recent studies have shown that similarity between autosomal and Y sequences can create perceived allelic differences between the sexes. However, Y duplications are most likely in species with large nonrecombining regions, in part because they simply represent larger targets for duplications. We assessed the genomes of 120 wild-caught guppies, which experience extensive predation- and pathogen-induced mortality and have a relatively small ancestral Y chromosome. We identified seven autosomal genes that show allelic differences between male and female adults. Five of these genes show clear evidence of whole or partial gene duplication between the Y chromosome and the autosomes. The remaining two genes show evidence of partial homology to the Y. Overall, our findings suggest that the guppy genome experiences a very low level of unresolved sexual conflict over survival, and instead the Y chromosome, despite its small ancestral size and recent origin, may nonetheless accumulate genes with male-specific functions.

A substantial amount of phenotypic diversity results from changes in gene expression levels and patterns. Understanding how the transcriptome evolves is therefore a key priority in identifying mechanisms of adaptive change. However, in contrast to powerful models of sequence evolution, we lack a consensus model of gene expression evolution. Furthermore, recent work has shown that many of the comparative approaches used to study gene expression are subject to biases that can lead to false signatures of selection. Here we first outline the main approaches for describing expression evolution and their inherent biases. Next, we bridge the gap between the fields of phylogenetic comparative methods and transcriptomics to reinforce the main pitfalls of inferring selection on expression patterns and use simulation studies to show that shifts in tissue composition can heavily bias inferences of selection. We close by highlighting the multi-dimensional nature of transcriptional variation and identifying major unanswered questions in disentangling how selection acts on the transcriptome. This paper examines the main approaches for studying gene expression evolution, tests their inherent biases and discusses open questions about the evolution of the transcriptome.

Males and females of the same species share the majority of their genomes, yet they are frequently exposed to conflicting selection pressures. Gene regulation is widely assumed to resolve these conflicting sex-specific selection pressures, and although there has been considerable focus on elucidating the role of gene expression level in sex-specific adaptation, other regulatory mechanisms have been overlooked. Alternative splicing enables different transcripts to be generated from the same gene, meaning that exons which have sex-specific beneficial effects can in theory be retained in the gene product, whereas exons with detrimental effects can be skipped. However, at present, little is known about how sex-specific selection acts on broad patterns of alternative splicing. Here, we investigate alternative splicing across males and females of multiple bird species. We identify hundreds of genes that have sex-specific patterns of splicing and establish that sex differences in splicing are correlated with phenotypic sex differences. Additionally, we find that alternatively spliced genes have evolved rapidly as a result of sex-specific selection and suggest that sex differences in splicing offer another route to sex-specific adaptation when gene expression level changes are limited by functional constraints. Overall, our results shed light on how a diverse transcriptional framework can give rise to the evolution of phenotypic sexual dimorphism.

A substantial amount of phenotypic diversity results from changes in gene regulation. Understanding how regulatory diversity evolves is therefore a key priority in identifying mechanisms of adaptive change. However, in contrast to powerful models of sequence evolution, we lack a consensus model of regulatory evolution. Furthermore, recent work has shown that many of the comparative approaches used to study gene regulation are subject to biases that can lead to false signatures of selection. In this review, we first outline the main approaches for describing regulatory evolution and their inherent biases. Next, we bridge the gap between the fields of comparative phylogenetic methods and transcriptomics to reinforce the main pitfalls of inferring regulatory selection and use simulation studies to show that shifts in tissue composition can heavily bias inferences of selection. We close by highlighting the multi-dimensional nature of regulatory variation and identifying major, unanswered questions in disentangling how selection acts on the transcriptome.

The sex chromosomes often follow unusual evolutionary trajectories. In particular, the sex-limited chromosomes frequently exhibit a small but unusual gene content in numerous species, where many genes have undergone massive gene amplification. The reasons for this remain elusive with a number of recent studies implicating meiotic drive, sperm competition, genetic drift, and gene conversion in the expansion of gene families. However, our understanding is primarily based on Y chromosome studies as few studies have systematically tested for copy number variation on W chromosomes. Here, we conduct a comprehensive investigation into the abundance, variability, and evolution of ampliconic genes on the avian W. First, we quantified gene copy number and variability across the duck W chromosome. We find a limited number of gene families as well as conservation in W-linked gene copy number across duck breeds, indicating that gene amplification may not be such a general feature of sex chromosome evolution as Y studies would initially suggest. Next, we investigated the evolution of HINTW, a prominent ampliconic gene family hypothesized to play a role in female reproduction and oogenesis. In particular, we investigated the factors driving the expansion of HINTW using contrasts between modern chicken and duck breeds selected for different female-specific selection regimes and their wild ancestors. Although we find the potential for selection related to fecundity in explaining small-scale gene amplification of HINTW in the chicken, purifying selection seems to be the dominant mode of evolution in the duck. Together, this challenges the assumption that HINTW is key for female fecundity across the avian phylogeny.

Sex chromosomes are typically comprised of a non-recombining region and a recombining pseudoautosomal region. Accurately quantifying the relative size of these regions is critical for sex-chromosome biology both from a functional and evolutionary perspective. The evolution of the pseudoautosomal boundary (PAB) is well documented in haplorrhines (apes and monkeys) but not in strepsirrhines (lemurs and lorises). Here, we studied the PAB of seven species representing the main strepsirrhine lineages by sequencing a male and a female genome in each species and using sex differences in coverage to identify the PAB. We found that during primate evolution, the PAB has remained unchanged in strepsirrhines whereas several recombination suppression events moved the PAB and shortened the pseudoautosomal region in haplorrhines. Strepsirrhines are well known to have much lower sexual dimorphism than haplorrhines. We suggest that mutations with antagonistic effects between males and females have driven recombination suppression and PAB evolution in haplorrhines

L13 ‘Moved’ seems a bit unspecific in this sentence, I would replace with extended PAR. P3 L12 I’m not aware of any direct evidence that recombination between the primate X and Y stopped instantaneously as ‘at once’ suggests. I would remove this. P3 L14 I am not sure what process is referred to in this sentence. P7 L12 I believe Wright et al showed extension of recombination suppression not additional strata P7 L17

The duration of the developmental period represents a fundamental axis of life-history variation, yet broad insights regarding the drivers of this diversity are currently lacking. Here, we test mechanistic and ecological explanations for the evolution of developmental duration using embryological data and information on incubation and fledging for 3096 avian species. Developmental phases associated primarily with growth are the longest and most variable, consistent with a role for allometric constraint in determining the duration of development. In addition, developmental durations retain a strong imprint of deep evolutionary history and body size differences among species explain less variation than previously thought. Finally, we reveal ecological correlates of developmental durations, including variables associated with the relative safety of the developmental environment and pressures of breeding phenology. Overall, our results provide broad-scale insight into the relative importance of mechanistic, ecological and evolutionary constraints in shaping the diversification of this key life-history trait.

We read with great interest the recent work in PNAS by Bergero et al. (1) describing differences in male and female recombination patterns on the guppy ( Poecilia reticulata ) sex chromosome. We fully agree that recombination in males is largely confined to the ends of the sex chromosome. Bergero et al. interpret these results to suggest that our previous findings of population-level variation in the degree of sex chromosome differentiation in this species (2) are incorrect. However, we suggest that their results are entirely consistent with our previous report, and that their interpretation presents a false controversy. Our population genomic results indicate that crossing over between the X and Y is rare across most of … [↵][1]1To whom correspondence may be addressed. Email: a.e.wright{at}sheffield.ac.uk. [1]: #xref-corresp-1-1

Once recombination is halted between the X and Y chromosomes, sex chromosomes begin to differentiate and transition to heteromorphism. While there is a remarkable variation across clades in the degree of sex chromosome divergence, far less is known about the variation in sex chromosome differentiation within clades. Here, we combined whole-genome and transcriptome sequencing data to characterize the structure and conservation of sex chromosome systems across Poeciliidae, the livebearing clade that includes guppies. We found that the Poecilia reticulata XY system is much older than previously thought, being shared not only with its sister species, Poecilia wingei, but also with Poecilia picta, which diverged roughly 20 million years ago. Despite the shared ancestry, we uncovered an extreme heterogeneity across these species in the proportion of the sex chromosome with suppressed recombination, and the degree of Y chromosome decay. The sex chromosomes in P. reticulata and P. wingei are largely homomorphic, with recombination in the former persisting over a substantial fraction. However, the sex chromosomes in P. picta are completely nonrecombining and strikingly heteromorphic. Remarkably, the profound degradation of the ancestral Y chromosome in P. picta is counterbalanced by the evolution of functional chromosome-wide dosage compensation in this species, which has not been previously observed in teleost fish. Our results offer important insight into the initial stages of sex chromosome evolution and dosage compensation.

One is a maximum likelihood (ML) approach to estimate haplotype frequencies between pairs of sites using an expectation maximization (EM) algorithm (Excoffier and Slatkin, 1995). From such estimated haplotype frequencies, we then calculate D, D′ and r for each pair of sites. This popular method has been previously used, for instance, to estimate ancestral LD from admixed populations from genotype data (Moltke et al., 2015). Here, we have adapted the latter method to a single unadmixed population and extended it to deal with genotype likelihoods rather than known genotypes. Following the notation in the original study (Moltke et al., 2015), let’s denote G = (G1, G2, ..., Gn) as the genotypes of n samples with Gi = (G 1 i , G 2 i ) being the genotypes of sample i for the pair of SNPs of interest. Likewise, we denote Di = (D 1 i , D 2 i ) as the sequencing reads data for sample i at the pair of SNPs. Lastly, let’s denote the frequency of haplotype j as pj, the unobserved pair of haplotypes for a sample as h = (h1, h2), and H as the set of all possible haplotypes. The likelihood for haplotype frequencies p = (pj) given the sequencing data D is given by:

There has been substantial interest of late in using population genetic methods to study sexual conflict, where an allele increases the fitness of one sex at some cost to the other (Mank, 2017). Population genomic scans for sexual conflict offer an important advance given the difficulties of identifying antagonistic alleles from more traditional methods, and could greatly increase our understanding of the extent and loci of sexual conflict. This is particularly true for studies in natural populations, for which obtaining accurate fitness measurements for each sex can be challenging. In this issue of Molecular Ecology, Bissegger, Laurentino, Roesti, and Berner (2019) present a cautionary tale about how to interpret these population genomic data.