Identifying homomorphic sex chromosomes from wild‐caught adults with limited genomic resources

We demonstrate a genotyping‐by‐sequencing approach to identify homomorphic sex chromosomes and their homolog in a distantly related reference genome, based on noninvasive sampling of wild‐caught individuals, in the moor frog Rana arvalis. Double‐digest RADseq libraries were generated using buccal swabs from 30 males and 21 females from the same population. Search for sex‐limited markers from the unfiltered data set (411 446 RAD tags) was more successful than searches from a filtered data set (33 073 RAD tags) for markers showing sex differences in heterozygosity or in allele frequencies. Altogether, we obtained 292 putatively sex‐linked RAD loci, 98% of which point to male heterogamety. We could map 15 of them to the Xenopus tropicalis genome, all but one on chromosome pair 1, which seems regularly co‐opted for sex determination among amphibians. The most efficient mapping strategy was a three‐step hierarchical approach, where R. arvalis reads were first mapped to a low‐coverage genome of Rana temporaria (17 My divergence), then the R. temporaria scaffolds to the Nanorana parkeri genome (90 My divergence), and finally the N. parkeri scaffolds to the X. tropicalis genome (210 My). We validated our conclusions with PCR primers amplifying part of Dmrt1, a candidate sex determination gene mapping to chromosome 1: a sex‐diagnostic allele was present in all 30 males but in none of the 21 females. Our approach is likely to be productive in many situations where biological samples and/or genomic resources are limited.     

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.     

Sex‐antagonistic genes,XY recombination and feminized Y chromosomes

The canonical model of sex‐chromosome evolution predicts that sex‐antagonistic (SA) genes play an instrumental role in the arrest of XY recombination and ensuing Y chromosome degeneration. Although this model might account for the highly differentiated sex chromosomes of birds and mammals, it does not fit the situation of many lineages of fish, amphibians or nonavian reptiles, where sex chromosomes are maintained homomorphic through occasional XY recombination and/or high turnover rates. Such situations call for alternative explanatory frameworks. A crucial issue at stake is the effect of XY recombination on the dynamics of SA genes and deleterious mutations. Using individual‐based simulations, we show that a complete arrest of XY recombination actually benefits females, not males. Male fitness is maximized at different XY recombination rates depending on SA selection, but never at zero XY recombination. This should consistently favour some level of XY recombination, which in turn generates a recombination load at sex‐linked SA genes. Hill–Robertson interferences with deleterious mutations also impede the differentiation of sex‐linked SA genes, to the point that males may actually fix feminized phenotypes when SA selection and XY recombination are low. We argue that sex chromosomes might not be a good localization for SA genes, and sex conflicts seem better solved through the differential expression of autosomal genes.     

Isolation and development of a molecular sex marker for <Emphasis Type="Italic">Bassiana duperreyi</Emphasis>, a lizard with XX/XY sex chromosomes and temperature-induced sex reversal

Sex determination in the endemic Australian lizard Bassiana duperreyi (Scincidae) is influenced by sex chromosomes and incubation temperature, challenging the traditional dichotomy in reptilian sex determination. Analysis of those interactions requires sex chromosome markers to identify temperature-induced sex reversal. Here, we report the isolation of Y chromosome DNA sequence from B. duperreyi using amplified fragment length polymorphism PCR, the conversion of that sequence to a single-locus assay, and its combination with a single-copy nuclear gene (C-mos) to form a duplex PCR test for chromosomal sex. The accuracy of the assay was tested on an independent panel of individuals with known phenotypic sex. When used on offspring from field nests, our test identified the likely occurrence of a low rate of natural sex reversal in this species. This work represents the first report of Y chromosome sequence from a reptile and one of the few reptile sex tests.     

Trans‐species variation in Dmrt1 is associated with sex determination in four European tree‐frog species

Empirical studies on the relative roles of occasional XY recombination versus sex‐chromosome turnover in preventing sex‐chromosome differentiation may shed light on the evolutionary forces acting on sex‐determination systems. Signatures of XY recombination are difficult to distinguish from those of homologous transitions (i.e., transitions in sex‐determination systems that keep sex‐chromosome identity): both models predict X and Y alleles at sex‐linked genes to cluster by species. However, the XY‐recombination model specifically predicts the reverse pattern (clustering by gametologs) for those genes that are directly involved in sex determination. Hence, the latter model can only be validated by identification of an ancestral sex‐determining region (SDR) with trans‐species polymorphism associated to sex. Here we combine a candidate‐gene approach with a genome scan to identify a small SDR shared by four species of a monophyletic clade of European tree frogs. This SDR encompasses at least the N‐terminal part of Dmrt1 and immediate upstream sequences. Our findings provide definitive evidence that sex‐chromosome homomorphy in this clade results only from XY recombination, and take an important step toward the identification of the sex‐determining locus. Moreover, the sex‐diagnostic markers we identify will enable research on environmental sex reversal in a wider range of frog species.     

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).     

A reciprocal translocation radically reshapes sex‐linked inheritance in the common frog

X and Y chromosomes can diverge when rearrangements block recombination between them. Here we present the first genomic view of a reciprocal translocation that causes two physically unconnected pairs of chromosomes to be coinherited as sex chromosomes. In a population of the common frog (Rana temporaria), both pairs of X and Y chromosomes show extensive sequence differentiation, but not degeneration of the Y chromosomes. A new method based on gene trees shows both chromosomes are sex‐linked. Furthermore, the gene trees from the two Y chromosomes have identical topologies, showing they have been coinherited since the reciprocal translocation occurred. Reciprocal translocations can thus reshape sex linkage on a much greater scale compared with inversions, the type of rearrangement that is much better known in sex chromosome evolution, and they can greatly amplify the power of sexually antagonistic selection to drive genomic rearrangement. Two more populations show evidence of other rearrangements, suggesting that this species has unprecedented structural polymorphism in its sex chromosomes.     

Genetic diversity,population structure and sex‐biased dispersal in three co‐evolving species

Genetic diversity and spatial structure of populations are important for antagonistic coevolution. We investigated genetic variation and population structure of three closely related European ant species: the social parasite Harpagoxenus sublaevis and its two host species Leptothorax acervorum and Leptothorax muscorum. We sampled populations in 12 countries and analysed eight microsatellite loci and an mtDNA sequence. We found high levels of genetic variation in all three species, only slightly less variation in the host L. muscorum. Using a newly introduced measure of differentiation (Jost’s D_est ), we detected strong population structuring in all species and less male‐biased dispersal than previously thought. We found no phylogeographic patterns that could give information on post‐glacial colonization routes – northern populations are as variable as more southern populations. We conclude that conditions for Thompson’s geographic mosaic of coevolution are ideal in this system: all three species show ample genetic variation and strong population structure.     

Culture modifies expectations of kinship and sex‐biased dispersal patterns: A case study of patrilineality and patrilocality in tribal yemen

Studies of the impact of post‐marital residence patterns on the distribution of genetic variation within populations have returned conflicting results. These studies have generally examined genetic diversity within and between groups with different post‐marriage residence patterns. Here, we directly examine Y chromosome microsatellite variation in individuals carrying a chromosome in the same Y haplogroup. We analyze Y chromosome data from two samples of Yemeni males: a sample representing the entire country and a sample from a large highland village. Our results support a normative patrilocality in highland Yemeni tribal populations, but also suggest that patrilocality is violated often enough to break down the expected correlation of genetic and geographic distance. We propose that a great deal of variation in male dispersal distance distributions is subsumed under the “patrilocal” label and that few human societies are likely to realize the idealized male dispersal distribution expected under strict patrilocality. In addition, we found almost no specific correspondence between social kinship and genetic patriline at the level of the clan (large, extended patrilineal kinship group) within a large, highland Yemeni village. We discuss ethnographic accounts that offer several cultural practices that explain exceptions to patrilocality and means by which social kinship and genetic patriline may become disentangled. © 2013 Wiley Periodicals, Inc.     

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.     

A demographic model for sex ratio evolution and the effects of sex‐biased offspring costs

The evolution of the primary sex ratio, the proportion of male births in an individual's offspring production strategy, is a frequency‐dependent process that selects against the more common sex. Because reproduction is shaped by the entire life cycle, sex ratio theory would benefit from explicitly two‐sex models that include some form of life cycle structure. We present a demographic approach to sex ratio evolution that combines adaptive dynamics with nonlinear matrix population models. We also determine the evolutionary and convergence stability of singular strategies using matrix calculus. These methods allow the incorporation of any population structure, including multiple sexes and stages, into evolutionary projections. Using this framework, we compare how four different interpretations of sex‐biased offspring costs affect sex ratio evolution. We find that demographic differences affect evolutionary outcomes and that, contrary to prior belief, sex‐biased mortality after parental investment can bias the primary sex ratio (but not the corresponding reproductive value ratio). These results differ qualitatively from the widely held conclusions of previous models that neglect demographic structure.     

The evolution of sex‐determining mechanisms: lessons from temperature‐sensitive mutations in sex determination genes in Caenorhabditis elegans

Sexual reproduction is one of the most taxonomically conserved traits, yet sex‐determining mechanisms (SDMs) are quite diverse. For instance, there are numerous forms of environmental sex determination (ESD), in which an organism’s sex is determined not by genotype, but by environmental factors during development. Important questions remain regarding transitions between SDMs, in part because the organisms exhibiting unique mechanisms often make difficult study organisms. One potential solution is to utilize mutant strains in model organisms better suited to answering these questions. We have characterized two such strains of the model nematode Caenorhabditis elegans. These strains harbour temperature‐sensitive mutations in key sex‐determining genes. We show that they display a sex ratio reaction norm in response to rearing temperature similar to other organisms with ESD. Next, we show that these mutations also cause deleterious pleiotropic effects on overall fitness. Finally, we show that these mutations are fundamentally different at the genetic sequence level. These strains will be a useful complement to naturally occurring taxa with ESD in future research examining the molecular basis of and the selective forces driving evolutionary transitions between sex determination mechanisms.     

Establishment of a strain inheriting a sex-linked SNP marker in Patagonian pejerrey (Odontesthes hatcheri), a species with both genotypic and temperature-dependent sex determination

The Patagonian pejerrey Odontesthes hatcheri is an atherinopsid species presenting genotypic sex determination (GSD) at intermediate temperatures and temperature-dependent sex determination at the low and high ranges of thermal tolerance. A recent study revealed the presence of a sex-linked SNP marker in some males of this species, but a strain which inherits the marker faithfully has not been established. This research was conducted to develop such a strain, for use as a tool to study the molecular mechanisms of gonadal sex differentiation and sexual dimorphism, and to obtain basic information on the GSD mode in this species. For these purposes, we performed backcrosses and full-sibling crosses using males and females whose presumptive genotypic sex was inferred from the presence of the sex-linked SNP marker. Four backcrosses between SNP⁻ daughters and their SNP⁺ father generated balanced sex ratios with the phenotypic sex matching the genotypic sex in most cases (98.21%) at an intermediate, sexually neutral temperature (21 °C). Full-sibling crosses between these four SNP⁻ females and their SNP⁺ brothers produced three progenies with balanced sex ratios and one with 94.4% males. The results of this study confirm that a strain inheriting the sex-linked SNP marker was successfully developed. Moreover, the inheritance pattern of the marker and the sex ratios of the progenies provide strong evidence that the GSD mode in O. hatcheri is the XX–XY system.     

Genetic sex assignment in wild populations using genotyping‐by‐sequencing data: A statistical threshold approach

Establishing the sex of individuals in wild systems can be challenging and often requires genetic testing. Genotyping‐by‐sequencing (GBS) and other reduced‐representation DNA sequencing (RRS) protocols (e.g., RADseq, ddRAD) have enabled the analysis of genetic data on an unprecedented scale. Here, we present a novel approach for the discovery and statistical validation of sex‐specific loci in GBS data sets. We used GBS to genotype 166 New Zealand fur seals (NZFS, Arctocephalus forsteri) of known sex. We retained monomorphic loci as potential sex‐specific markers in the locus discovery phase. We then used (i) a sex‐specific locus threshold (SSLT) to identify significantly male‐specific loci within our data set; and (ii) a significant sex‐assignment threshold (SSAT) to confidently assign sex in silico the presence or absence of significantly male‐specific loci to individuals in our data set treated as unknowns (98.9% accuracy for females; 95.8% for males, estimated via cross‐validation). Furthermore, we assigned sex to 86 individuals of true unknown sex using our SSAT and assessed the effect of SSLT adjustments on these assignments. From 90 verified sex‐specific loci, we developed a panel of three sex‐specific PCR primers that we used to ascertain sex independently of our GBS data, which we show amplify reliably in at least two other pinniped species. Using monomorphic loci normally discarded from large SNP data sets is an effective way to identify robust sex‐linked markers for nonmodel species. Our novel pipeline can be used to identify and statistically validate monomorphic and polymorphic sex‐specific markers across a range of species and RRS data sets.     

Genetic diversity and sex‐bias dispersal of plateau pika in Tibetan plateau

Dispersal is an important aspect in organism's life history which could influence the rate and outcome of evolution of organism. Plateau pika is the keystone species in community of grasslands in Tibetan Plateau. In this study, we combine genetic and field data to character the population genetic pattern and dispersal dynamics in plateau pika (Ochotona curzoniae). Totally, 1,352 individual samples were collected, and 10 microsatellite loci were analyzed. Results revealed that plateau pika possessed high genetic diversity and inbreeding coefficient in a fine‐scale population. Dispersal distance is short and restricted in about 20 m. An effective sex‐biased dispersal strategy is employed by plateau pika: males disperse in breeding period for mating while females do it after reproduction for offspring and resource. Inbreeding avoiding was shown as the common driving force of dispersal, together with the other two factors, environment and resource. In addition, natal dispersal is female biased. More detailed genetic analyzes are needed to confirm the role of inbreeding avoidance and resource competition as ultimate cause of dispersal patterns in plateau pika.     

Biased sex‐ratio and sex‐biased heterozygote disadvantage affect the maintenance of a genetic polymorphism and the properties of hybrid zones

The evolution of biodiversity is a major issue of modern biology, and it is becoming increasingly topical as the ongoing erosion of diversity puts serious threats on human well‐being. An elementary mechanism that allows maintaining diversity is the interplay between dispersal and heterozygote selective disadvantage, which can lead to self‐sustainable spatial genetic structures and is central to the stability of hybrid zones. Theoretical studies supporting the importance of this mechanism assume a balanced sex‐ratio and a heterozygote disadvantage equally affecting both sexes, despite the multiplicity of empirical evidence that (i) adult sex‐ratio is usually biased towards either male or female and that (ii) heterozygote disadvantage often affects a single sex. We expanded the existing theory by weighting the strength of selection against heterozygote according to the biased in sex‐ratio and in heterozygote disadvantage. The range of conditions allowing for the maintenance of polymorphism can then either double or vanish. We discuss the implications of such finding for birds, mammals and insects diversity. Finally, we provide simple analytical predictions about the effect of those biased on the width and speed of hybrid zones and on the time for the spread of beneficial mutations through such zones.     

The paternal‐sex‐ratio (PSR) chromosome in natural populations of Nasonia (Hymenoptera: Chalcidoidea)

Selfish genetic elements may be important in promoting evolutionary change. Paternal sex ratio (PSR) is a selfish B chromosome that causes all‐male families in the haplodiploid parasitic wasp Nasonia vitripennis, by inducing paternal genome loss in fertilized eggs. The natural distribution and frequency of this chromosome in North American populations of N. vitripennis was investigated using a combination of phenotypic and molecular assays. Sampling throughout North America failed to recover PSR except from populations in the Great Basin area of western North America. Extensive sampling of Great Basin populations revealed PSR in frequencies ranging from 0 to 6% at different collection sites, and extended its distribution to Idaho and Wyoming. Intensive sampling in upstate New York did not detect the chromosome. Frequencies of the maternal‐sex ratio distorter (MSR), son killer (SK) and virgin females ranged from 0 to 12%. Paternal sex ratio may be restricted to the Great Basin because its spread is hampered by geographical barriers, or because populations in other areas are not conducive to PSR maintenance. However, it cannot be ruled out that PSR occurs in other regions at very low frequencies. The apparent limited distribution and low frequency of PSR suggest that it will have relatively little impact on genome evolution in Nasonia.     

Isolation of highly polymorphic autosomal microsatellite loci and a sex‐linked locus from sugarbirds

We describe the isolation of six tetranucleotide microsatellites from the Cape sugarbird (Promerops cafer) using an enrichment protocol. All loci were highly variable with number of alleles ranging from nine to 26 and values of observed heterozygosity ranging from 0.534 to 0.931. All loci were in Hardy–Weinberg equilibrium with the exception of Pro66 and Pro86. Further analysis of Pro86 indicated it was Z‐linked. All loci amplified and were variable in the congeneric Gurneys sugarbird (Promerops gurneyi). These loci will be used in mating system and phylogeographic studies of Cape sugarbirds in South Africa.     

Geographic variation in sex‐chromosome differentiation in the common frog (Rana temporaria)

In sharp contrast with birds and mammals, sex‐determination systems in ectothermic vertebrates are often highly dynamic and sometimes multifactorial. Both environmental and genetic effects have been documented in common frogs (Rana temporaria). One genetic linkage group, mapping to the largest pair of chromosomes and harbouring the candidate sex‐determining gene Dmrt1, associates with sex in several populations throughout Europe, but association varies both within and among populations. Here, we show that sex association at this linkage group differs among populations along a 1500‐km transect across Sweden. Genetic differentiation between sexes is strongest (F_ST = 0.152) in a northern‐boreal population, where male‐specific alleles and heterozygote excesses (F_IS = ?0.418 in males, +0.025 in females) testify to a male‐heterogametic system and lack of X‐Y recombination. In the southernmost population (nemoral climate), in contrast, sexes share the same alleles at the same frequencies (F_ST = 0.007 between sexes), suggesting unrestricted recombination. Other populations show intermediate levels of sex differentiation, with males falling in two categories: some cluster with females, while others display male‐specific Y haplotypes. This polymorphism may result from differences between populations in the patterns of X‐Y recombination, co‐option of an alternative sex‐chromosome pair, or a mixed sex‐determination system where maleness is controlled either by genes or by environment depending on populations or families. We propose approaches to test among these alternative models, to disentangle the effects of climate and phylogeography on the latitudinal trend, and to sort out how this polymorphism relates to the ‘sexual races’ described in common frogs in the 1930s.