High rates of male courtship in a female-ornamented pipefish.

In species with sex-specific signalling traits that appear to be ornamental (i.e. are conspicuous and with no obvious natural selection benefit), the ornamented sex typically initiates courtship and is most active in courtship. Here, we report for the first time courtship displays in the extremely sexually dimorphic, female-ornamented wide-bodied pipefish (Stigmatopora nigra), revealing unexpected behaviours. Females use their sex-specific ornament during courtship displays, as expected, but rarely in female-female interactions. Surprisingly, males initiated 61% of reciprocated courtship bouts and chased females in 17% of the bouts. This chasing behaviour could be a form of male harassment or be indicative of female disinterest in ardent males, either of which was unexpected to be found in this female-ornamented species. Our results highlight the need to study the details of species' behaviours in considering the potential roles of sexual selection and sexual conflict in shaping sexual dimorphism.

The roles of sexual selection and sexual conflict in shaping patterns of genome and transcriptome variation.

Sexual dimorphism is one of the most prevalent, and often the most extreme, examples of phenotypic variation within species, and arises primarily from genomic variation that is shared between females and males. Many sexual dimorphisms arise through sex differences in gene expression, and sex-biased expression is one way that a single, shared genome can generate multiple, distinct phenotypes. Although many sexual dimorphisms are expected to result from sexual selection, and many studies have invoked the possible role of sexual selection to explain sex-specific traits, the role of sexual selection in the evolution of sexually dimorphic gene expression remains difficult to differentiate from other forms of sex-specific selection. In this Review, we propose a holistic framework for the study of sex-specific selection and transcriptome evolution. We advocate for a comparative approach, across tissues, developmental stages and species, which incorporates an understanding of the molecular mechanisms, including genomic variation and structure, governing gene expression. Such an approach is expected to yield substantial insights into the evolution of genetic variation and have important applications in a variety of fields, including ecology, evolution and behaviour.

The evolution of the testis transcriptome in pregnant male pipefishes and seahorses.

In many animals, sperm competition and sexual conflict are thought to drive the rapid evolution of male-specific genes, especially those expressed in the testes. A potential exception occurs in the male pregnant pipefishes, where females transfer eggs to the males, eliminating testes from participating in these processes. Here, we show that testis-related genes differ dramatically in their rates of molecular evolution and expression patterns in pipefishes and seahorses (Syngnathidae) compared to other fish. Genes involved in testis or sperm function within syngnathids experience weaker selection in comparison to their orthologs in spawning and livebearing fishes. An assessment of gene turnover and expression in the testis transcriptome suggests that syngnathids have lost (or significantly reduced expression of) important classes of genes from their testis transcriptomes compared to other fish. Our results indicate that more than 50 million years of male pregnancy have removed syngnathid testes from the molecular arms race that drives the rapid evolution of male reproductive genes in other taxa.

The population genomics of repeated freshwater colonizations by Gulf pipefish.

How organisms adapt to the novel challenges imposed by the colonization of a new habitat has long been a central question in evolutionary biology. When multiple populations of the same species independently adapt to similar environmental challenges, the question becomes whether the populations have arrived at their adaptations through the same genetic mechanisms. In recent years, genetic techniques have been used to tackle these questions by investigating the genome-level changes underlying local adaptation. Here, we present a genomic analysis of colonization of freshwater habitats by a primarily marine fish, the Gulf pipefish (Syngnathus scovelli). We sample pipefish from four geographically distinct freshwater locations and use double-digest restriction site associated DNA sequencing to compare them to 12 previously studied saltwater populations. The two most geographically distant and isolated freshwater populations are the most genetically distinct, although demographic analysis suggests that these populations are experiencing ongoing migration with their saltwater neighbours. Additionally, outlier regions were found genome-wide, showing parallelism across ecotype pairs. We conclude that these multiple freshwater colonizations involve similar genomic regions, despite the large geographical distances and different underlying mechanisms. These similar patterns are probably facilitated by the interacting effects of intrinsic barriers, gene flow among populations and ecological selection in the Gulf pipefish.

The Estrogen-Responsive Transcriptome of Female Secondary Sexual Traits in the Gulf Pipefish.

Sexual dimorphism often results from hormonally regulated trait differences between the sexes. In sex-role-reversed vertebrates, females often have ornaments used in mating competition that are expected to be under hormonal control. Males of the sex-role-reversed Gulf pipefish (Syngnathus scovelli) develop female-typical traits when they are exposed to estrogens. We aimed to identify genes whose expression levels changed during the development and maintenance of female-specific ornaments. We performed RNA-sequencing on skin and muscle tissue in male Gulf pipefish with and without exposure to estrogen to investigate the transcriptome of the sexually dimorphic ornament of vertical iridescent bands found in females and estrogen-exposed males. We further compared differential gene expression patterns between males and females to generate a list of genes putatively involved in the female secondary sex traits of bands and body depth. A detailed analysis of estrogen-receptor binding sites demonstrates that estrogen-regulated genes tend to have nearby cis-regulatory elements. Our results identified a number of genes that differed between the sexes and confirmed that many of these were estrogen-responsive. These estrogen-regulated genes may be involved in the arrangement of chromatophores for color patterning, as well as in the growth of muscles to achieve the greater body depth typical of females in this species. In addition, anaerobic respiration and adipose tissue could be involved in the rigors of female courtship and mating competition. Overall, this study generates a number of interesting hypotheses regarding the genetic basis of a female ornament in a sex-role-reversed pipefish.

The why, when, and how of computing in biology classrooms.

Many biologists are interested in teaching computing skills or using computing in the classroom, despite not being formally trained in these skills themselves. Thus biologists may find themselves researching how to teach these skills, and therefore many individuals are individually attempting to discover resources and methods to do so. Recent years have seen an expansion of new technologies to assist in delivering course content interactively. Educational research provides insights into how learners absorb and process information during interactive learning. In this review, we discuss the value of teaching foundational computing skills to biologists, and strategies and tools to do so. Additionally, we review the literature on teaching practices to support the development of these skills. We pay special attention to meeting the needs of diverse learners, and consider how different ways of delivering course content can be leveraged to provide a more inclusive classroom experience. Our goal is to enable biologists to teach computational skills and use computing in the classroom successfully.

The future of parentage analysis: From microsatellites to SNPs and beyond.

Parentage analysis is a cornerstone of molecular ecology that has delivered fundamental insights into behaviour, ecology and evolution. Microsatellite markers have long been the king of parentage, their hypervariable nature conferring sufficient power to correctly assign offspring to parents. However, microsatellite markers have seen a sharp decline in use with the rise of next-generation sequencing technologies, especially in the study of population genetics and local adaptation. The time is ripe to review the current state of parentage analysis and see how it stands to be affected by the emergence of next-generation sequencing approaches. We find that single nucleotide polymorphisms (SNPs), the typical next-generation sequencing marker, remain underutilized in parentage analysis but are gaining momentum, with 58 SNP-based parentage analyses published thus far. Many of these papers, particularly the earlier ones, compare the power of SNPs and microsatellites in a parentage context. In virtually every case, SNPs are at least as powerful as microsatellite markers. As few as 100-500 SNPs are sufficient to resolve parentage completely in most situations. We also provide an overview of the analytical programs that are commonly used and compatible with SNP data. As the next-generation parentage enterprise grows, a reliance on likelihood and Bayesian approaches, as opposed to strict exclusion, will become increasingly important. We discuss some of the caveats surrounding the use of next-generation sequencing data for parentage analysis and conclude that the future is bright for this important realm of molecular ecology.

Guidelines for planning genomic assessment and monitoring of locally adaptive variation to inform species conservation.

Identifying and monitoring locally adaptive genetic variation can have direct utility for conserving species at risk, especially when management may include actions such as translocations for restoration, genetic rescue, or assisted gene flow. However, genomic studies of local adaptation require careful planning to be successful, and in some cases may not be a worthwhile use of resources. Here, we offer an adaptive management framework to help conservation biologists and managers decide when genomics is likely to be effective in detecting local adaptation, and how to plan assessment and monitoring of adaptive variation to address conservation objectives. Studies of adaptive variation using genomic tools will inform conservation actions in many cases, including applications such as assisted gene flow and identifying conservation units. In others, assessing genetic diversity, inbreeding, and demographics using selectively neutral genetic markers may be most useful. And in some cases, local adaptation may be assessed more efficiently using alternative approaches such as common garden experiments. Here, we identify key considerations of genomics studies of locally adaptive variation, provide a road map for successful collaborations with genomics experts including key issues for study design and data analysis, and offer guidelines for interpreting and using results from genomic assessments to inform monitoring programs and conservation actions.

Substantial differences in bias between single-digest and double-digest RAD-seq libraries: A case study.

The trade-offs of using single-digest vs. double-digest restriction site-associated DNA sequencing (RAD-seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD-seq. Sixty individuals were subjected to single-digest RAD-seq (sdRAD-seq), and the remaining 384 individuals were genotyped using a double-digest RAD-seq (ddRAD-seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD-seq than with ddRAD-seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD-seq versus ddRAD-seq. Our finding that sdRAD-seq and ddRAD-seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations.

Constraints on the FST-Heterozygosity Outlier Approach.

The FST-heterozygosity outlier approach has been a popular method for identifying loci under balancing and positive selection since Beaumont and Nichols first proposed it in 1996 and recommended its use for studies sampling a large number of independent populations (at least 10). Since then, their program FDIST2 and a user-friendly program optimized for large datasets, LOSITAN, have been used widely in the population genetics literature, often without the requisite number of samples. We observed empirical datasets whose distributions could not be reconciled with the confidence intervals generated by the null coalescent island model. Here, we use forward-in-time simulations to investigate circumstances under which the FST-heterozygosity outlier approach performs poorly for next-generation single nucleotide polymorphism (SNP) datasets. Our results show that samples involving few independent populations, particularly when migration rates are low, result in distributions of the FST-heterozygosity relationship that are not described by the null model implemented in LOSITAN. In addition, even under favorable conditions LOSITAN rarely provides confidence intervals that precisely fit SNP data, making the associated P-values only roughly valid at best. We present an alternative method, implemented in a new R package named fsthet, which uses the raw empirical data to generate smoothed outlier plots for the FST-heterozygosity relationship.

Genome-wide selection components analysis in a fish with male pregnancy.

A major goal of evolutionary biology is to identify the genome-level targets of natural and sexual selection. With the advent of next-generation sequencing, whole-genome selection components analysis provides a promising avenue in the search for loci affected by selection in nature. Here, we implement a genome-wide selection components analysis in the sex role reversed Gulf pipefish, Syngnathus scovelli. Our approach involves a double-digest restriction-site associated DNA sequencing (ddRAD-seq) technique, applied to adult females, nonpregnant males, pregnant males, and their offspring. An FST comparison of allele frequencies among these groups reveals 47 genomic regions putatively experiencing sexual selection, as well as 468 regions showing a signature of differential viability selection between males and females. A complementary likelihood ratio test identifies similar patterns in the data as the FST analysis. Sexual selection and viability selection both tend to favor the rare alleles in the population. Ultimately, we conclude that genome-wide selection components analysis can be a useful tool to complement other approaches in the effort to pinpoint genome-level targets of selection in the wild.

Population genomics reveals multiple drivers of population differentiation in a sex-role-reversed pipefish.

A major goal of molecular ecology is to identify the causes of genetic and phenotypic differentiation among populations. Population genomics is suitably poised to tackle these key questions by diagnosing the evolutionary mechanisms driving divergence in nature. Here, we set out to investigate the evolutionary processes underlying population differentiation in the Gulf pipefish, Syngnathus scovelli. We sampled approximately 50 fish from each of 12 populations distributed from the Gulf coast of Texas to the Atlantic coast of Florida and performed restriction-site-associated DNA sequencing to identify SNPs throughout the genome. After imposing quality and stringency filters, we selected a panel of 6348 SNPs present in all 12 populations, 1753 of which were not physically linked. We identified a genome-wide pattern of isolation by distance, in addition to a more substantial genetic break separating populations in the Gulf of Mexico from those in the Atlantic. We also used several divergence outlier approaches and tests for genotype-environment correlations to identify 400 SNPs putatively involved in local adaptation. Patterns of phenotypic differentiation and variation diverged from the overall genomic pattern, suggesting that selection, phenotypic plasticity or demographic factors may be shaping phenotypes in distinct populations. Overall, our results suggest that population divergence is driven by a variety of factors in S. scovelli, including neutral processes and selection on multiple traits.

The Effects of Synthetic Estrogen Exposure on the Sexually Dimorphic Liver Transcriptome of the Sex-Role-Reversed Gulf Pipefish.

Species exhibiting sex-role reversal provide an unusual perspective on the evolution of sex roles and sex differences. However, the proximate effects of sex-role reversal are largely unknown. Endocrine disruptors provide an experimental mechanism to address hormonal regulation of sexually dimorphic gene expression in sex-role-reversed taxa. Here, we investigate gene expression patterns in the liver of the sex-role-reversed Gulf pipefish, because the liver is known to be sexually dimorphic and estrogen-regulated in species with conventional sex roles. Using next-generation RNA-sequencing technology (RNA-seq), we detected sexually dimorphic hepatic gene expression patterns, with a total of 482 differentially expressed genes between the sexes in Gulf pipefish. Two-thirds of these genes were over-expressed in females, and the sex-specific transcriptomes of this sex-role-reversed pipefish's liver were superficially similar to those of fishes with conventional sex-roles. We exposed females, pregnant males, and non-pregnant males to 17α-ethinylestradiol (EE2) at ecologically relevant concentrations of 5ng/L and compared gene expression patterns in the livers of exposed fish to control fish. Several genes that were up-regulated in EE2-exposed males relative to control males were also found to be female-biased in control animals. These genes included several of the classic estrogen biomarkers, such as vitellogenin, choriogenin, and zona pellucida. Thus, estrogen exposure induced feminization of the male liver transcriptome in a sex-role-reversed pipefish. These results suggest that the ancestral state of estrogen-regulated female reproductive physiology has been retained in all sex-role-reversed vertebrates thus far studied, despite substantial evolution of the hormonal regulation of ornamentation and mating behavior in these interesting taxa.

Identifying signatures of sexual selection using genomewide selection components analysis.

Sexual selection must affect the genome for it to have an evolutionary impact, yet signatures of selection remain elusive. Here we use an individual-based model to investigate the utility of genome-wide selection components analysis, which compares allele frequencies of individuals at different life history stages within a single population to detect selection without requiring a priori knowledge of traits under selection. We modeled a diploid, sexually reproducing population and introduced strong mate choice on a quantitative trait to simulate sexual selection. Genome-wide allele frequencies in adults and offspring were compared using weighted F ST values. The average number of outlier peaks (i.e., those with significantly large F ST values) with a quantitative trait locus in close proximity ("real" peaks) represented correct diagnoses of loci under selection, whereas peaks above the F ST significance threshold without a quantitative trait locus reflected spurious peaks. We found that, even with moderate sample sizes, signatures of strong sexual selection were detectable, but larger sample sizes improved detection rates. The model was better able to detect selection with more neutral markers, and when quantitative trait loci and neutral markers were distributed across multiple chromosomes. Although environmental variation decreased detection rates, the identification of real peaks nevertheless remained feasible. We also found that detection rates can be improved by sampling multiple populations experiencing similar selection regimes. In short, genome-wide selection components analysis is a challenging but feasible approach for the identification of regions of the genome under selection.