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Molecular evolutionary studies usually focus on genes with clear roles in adult fitness or on developmental genes expressed at multiple time points during the life of the organism. Here, we examine the evolutionary dynamics of Drosophila glue genes, a set of eight genes tasked with a singular primary function during a specific developmental stage: the production of glue that allows animal pupa to attach to a substrate for several days during metamorphosis. Using phenotypic assays and available data from transcriptomics, PacBio genomes, and genetic variation from global populations, we explore the selective forces acting on the glue genes within the cosmopolitan D. melanogaster species and its five closely related species, D. simulans, D. sechellia, D. mauritiana, D. yakuba, and D. teissieri. We observe a three-fold difference in glue adhesion between the least and the most adhesive D. melanogaster strain, indicating a strong genetic component to phenotypic variation. These eight glue genes are among the most highly expressed genes in salivary glands yet they display no notable codon bias. New copies of Sgs3 and Sgs7 are found in D. yakuba and D. teissieri with the Sgs3 coding sequence evolving rapidly after duplication in the D. yakuba branch. Multiple sites along the various glue genes appear to be constrained. Our population genetics analysis in D. melanogaster suggests signs of local adaptive evolution for Sgs3, Sgs5 and Sgs5bis and traces of selective sweeps for Sgs1, Sgs3, Sgs7 and Sgs8. Our work shows that stage-specific genes can be subjected to various dynamic evolutionary forces. (249 words)Significance statementDrosophila larvae produce a glue to stick themselves to a substrate for several days during metamorphosis. Here we observe wide variation in stickiness among Drosophila melanogaster strains and we analyze the molecular evolution of eight glue genes. We find several recent gene duplications and heterogenous rates of evolution among these genes.

Squamates include all lizards and snakes, and display some of the most diverse and extreme morphological adaptations among vertebrates. However, compared with birds and mammals, relatively few resources exist for comparative genomic analyses of squamates, hampering efforts to understand the molecular bases of phenotypic diversification in such a speciose clade. In particular, the ∼400 species of anole lizard represent an extensive squamate radiation. Here, we sequence and assemble the draft genomes of three anole species—Anolis frenatus, Anolis auratus, and Anolis apletophallus—for comparison with the available reference genome of Anolis carolinensis. Comparative analyses reveal a rapid background rate of molecular evolution consistent with a model of punctuated equilibrium, and strong purifying selection on functional genomic elements in anoles. We find evidence for accelerated evolution in genes involved in behavior, sensory perception, and reproduction, as well as in genes regulating limb bud development and hindlimb specification. Morphometric analyses of anole fore and hindlimbs corroborated these findings. We detect signatures of positive selection across several genes related to the development and regulation of the forebrain, hormones, and the iguanian lizard dewlap, suggesting molecular changes underlying behavioral adaptations known to reinforce species boundaries were a key component in the diversification of anole lizards.

Gene expression patterns assayed across development can offer key clues about a gene’s function and regulatory role. Drosophila melanogaster is ideal for such investigations as multiple individual and high-throughput efforts have captured the spatiotemporal patterns of thousands of embryonic expressed genes in the form of in situ images. FlyExpress (www.flyexpress.net), a knowledgebase based on a massive and unique digital library of standardized images and a simple search engine to find coexpressed genes, was created to facilitate the analytical and visual mining of these patterns. Here, we introduce the next generation of FlyExpress resources to facilitate the integrative analysis of sequence data and spatiotemporal patterns of expression from images. FlyExpress 7 now includes over 100,000 standardized in situ images and implements a more efficient, user-defined search algorithm to identify coexpressed genes via Genomewide Expression Maps (GEMs). Shared motifs found in the upstream 5′ regions of any pair of coexpressed genes can be visualized in an interactive dotplot. Additional webtools and link-outs to assist in the downstream validation of candidate motifs are also provided. Together, FlyExpress 7 represents our largest effort yet to accelerate discovery via the development and dispersal of new webtools that allow researchers to perform data-driven analyses of coexpression (image) and genomic (sequence) data.

Background Expression patterns between males and females vary in every adult tissue, even in organs with no conspicuous dimorphisms such as the heart. While studies of male and female differences have traditionally focused on the influence of sex hormones, these do not account for all the differences at the molecular and epigenetic levels. We previously reported that a substantial number of genes were differentially expressed in male and female mouse embryonic stem (ES) cells and revealed dose-dependent enhancer activity in response to Prdm14, a key pluripotency factor expressed more highly in female ES cells. In this work, we investigated the role of Prdm14 in establishing sex-specific gene expression networks. We surveyed the sex-specific landscape in early embryogenesis with special reference to cardiac development. We generated sex-specific co-expression networks from mouse ES cells, examined the presence of sex-specific chromatin domains, and analyzed previously published datasets from different developmental time points to characterize how sex-biased gene expression waxes and wanes to evaluate whether sex-biased networks are detectable throughout heart development. Results We performed ChIP-seq on male and female mouse ES cells to determine differences in chromatin status. Our study reveals sex-biased histone modifications, underscoring the potential for the sex chromosome complement to prime the genome differently in early development with consequences for later expression biases. Upon differentiation of ES cells to cardiac precursors, we found sex-biased expression of key transcription and epigenetic factors, some of which persisted from the undifferentiated state. Using network analyses, we also found that Prdm14 plays a prominent role in regulating a subset of dimorphic expression patterns. To determine whether sex-biased expression is present throughout cardiogenesis, we re-analyzed data from two published studies that sampled the transcriptomes of mouse hearts from 8.5 days post-coitum embryos to neonates and adults. We found sex-biased expression at every stage in heart development, and interestingly, identified a subset of genes that exhibit the same bias across multiple cardiogenic stages. Conclusions Overall, our results support the existence of sexually dimorphic gene expression profiles and regulatory networks at every stage of cardiac development, some of which may be established in early embryogenesis and epigenetically perpetuated. Electronic supplementary material The online version of this article (10.1186/s13293-019-0259-1) contains supplementary material, which is available to authorized users.

BACKGROUND African Americans with head and neck squamous cell carcinoma (HNSCC) have a lower survival rate than whites. This study investigated the functional importance of ancestry-informative single-nucleotide polymorphisms (SNPs) in HNSCC and also examined the effect of functionally important genetic elements on racial disparities in HNSCC survival. METHODS Ancestry-informative SNPs, RNA sequencing, methylation, and copy number variation data for 316 oral cavity and laryngeal cancer patients were analyzed across 178 DNA repair genes. The results of expression quantitative trait locus (eQTL) analyses were also replicated with a Gene Expression Omnibus (GEO) data set. The effects of eQTLs on overall survival (OS) and disease-free survival (DFS) were evaluated. RESULTS Five ancestry-related SNPs were identified as cis-eQTLs in the DNA polymerase β (POLB) gene (false discovery rate [FDR]

Males and females differ across a broad spectrum of morphological, physiological, and behavioral characters. In fact, sexually dimorphic traits typically contribute the largest component of phenotypic variance in most taxa that use sex to reproduce. However, we know very little about the mechanisms that maintain these dimorphic states and how these sexually dimorphic traits evolve. Here, we review our current knowledge of the underlying genetic basis of sexual dimorphism in humans. First, we briefly review the etiology of sex differences starting from sex determination's initial switch early in embryogenesis. We then survey recent sex-biased transcriptomic expression literature in order to provide additional insight into the landscape of sex-biased gene expression in both gonadal and non-gonadal tissues: from overall prevalence to tissue specificity to conservation across species. Finally, we discuss implications of sex-biased genetic architecture to human health and disease in light of the National Institute of Health's recently proposed initiative to promote study samples from both sexes.

A large portion of the annotated genes in Drosophila melanogaster show sex-biased expression, indicating that sex and reproduction-related genes (SRR genes) represent an appreciable component of the genome. Previous studies, in which subsets of genes were compared among few Drosophila species, have found that SRR genes exhibit unusual evolutionary patterns. Here, we have used the newly released genome sequences from 12 Drosophila species, coupled to a larger set of SRR genes, to comprehensively test the generality of these patterns. Among 2505 SRR genes examined, including ESTs with biased expression in reproductive tissues and genes characterized as involved in gametogenesis, we find that a relatively high proportion of SRR genes have experienced accelerated divergence throughout the genus Drosophila. Several testis-specific genes, male seminal fluid proteins (SFPs), and spermatogenesis genes show lineage-specific bursts of accelerated evolution and positive selection. SFP genes also show evidence of lineage-specific gene loss and/or gain. These results bring us closer to understanding the details of the evolutionary dynamics of SRR genes with respect to species divergence.

Among closely related taxa, proteins involved in reproduction generally evolve more rapidly than other proteins. Here, we apply a functional and comparative genomics approach to compare functional divergence across a deep phylogenetic array of egg-laying and live-bearing vertebrate taxa. We aligned and annotated a set of 4,986 1 : 1 : 1 : 1 : 1 orthologs inAnolis carolinensis(green lizard),Danio rerio(zebrafish),Xenopus tropicalis(frog),Gallus gallus(chicken), andMus musculus(mouse) according to function using ESTs from available reproductive (including testis and ovary) and non-reproductive tissues as well as Gene Ontology. For each species lineage, genes were further classified as tissue-specific (found in a single tissue) or tissue-expressed (found in multiple tissues). Within independent vertebrate lineages, we generally find that gonadal-specific genes evolve at a faster rate than gonadal-expressed genes and significantly faster than non-reproductive genes. Among the gonadal set, testis genes are generally more diverged than ovary genes. Surprisingly, an opposite but nonsignificant pattern is found among the subset of orthologs that remained functionally conserved acrossallfive lineages. These contrasting evolutionary patterns found between functionally diverged and functionally conserved reproductive orthologs provide evidence for pervasive and potentially cryptic lineage-specific selective processes on ancestral reproductive systems in vertebrates.

Strawberry (Fragaria spp.) is a valuable fruit crop as well as an outstanding system for studying functional genomics in plants. The goal of this study was to substantially increase and analyze the available expressed sequence information in the genus by examining the transcriptome of the cultivated strawberry (Fragaria × ananassa Duchesne). To maximize transcript diversity and discovery, plants representing an octoploid strawberry cultivar were subjected to a broad range of treatments. Plant materials were pooled by tissue type. cDNA pools were sequenced by the Roche-454 GS-FLX system and assembled into over 32,000 contigs. Predictions of cellular localization and function were made by associating assembled contigs to annotated homologs, and the tissue pool tags provided a means to assess the overall expression pattern for any given transcript. Contigs comprised of reads originating from only one organ type and those present equally in all plant organs were both identifi ed. Bacterial and fungal sequences found in the strawberry samples provide a metagenomic survey of the microbial community of a greenhouse strawberry plant. This study utilized an innovative assembly strategy on pooled tissues, thus providing a foundation for developing tissue-specifi c tools, an opportunity to identify alleles for marker-assisted selection, a reference of strawberry gene annotations, and a basis for comparative transcriptomics between cultivated strawberry, its diploid ancestors, and the wider Rosaceae family. C ULTIVATED STRAWBERRY is a valuable crop, prized for its fl avorful and nutritious fruits. Strawberry is an important crop for many regional economies worldwide and off ers high levels of healthful compounds. Species within the genus Fragaria occur naturally throughout the northern hemisphere and represent many levels of ploidy, including wild diploids and the wild and cultivated octoploids (reviewed in Folta and Davis, 2006; Hummer and Hancock, 2009). Recent attention has been brought to the diploid strawberry species Fragaria vesca Coville because of its utility as a highly tractable experimental system within the economically important Rosaceae family (Folta and Davis, 2006). Strawberry is emerging as a powerful functional genomics system. Fragaria vesca’s haploid genome size of ~200 Mb is among the smallest of the rosaceous crop plants (Shulaev et al., 2008), and its complete genome sequence will be available in 2010, providing an invaluable resource with reference to its octoploid, cultivated relatives. Most importantly, several genotypes of both diploid and octoploid strawberry are readily transformable, making it an outstanding system for functional studies. Protocols exist for diploid and octoploid lines that allow effi cient regeneration of transgenic materials in a period of weeks to months (Folta and Dhingra, 2006; Mezzetti and

Short-read sequencing techniques provide the opportunity to capture genome-wide sequence data in a single experiment. A current challenge is to identify questions that shallow-depth genomic data can address successfully and to develop corresponding analytical methods that are statistically sound. Here, we apply the Roche/454 platform to survey natural variation in strains of Drosophila melanogaster from an African (n = 3) and a North American (n = 6) population. Reads were aligned to the reference D. melanogaster genomic assembly, single nucleotide polymorphisms were identified, and nucleotide variation was quantified genome wide. Simulations and empirical results suggest that nucleotide diversity can be accurately estimated from sparse data with as little as 0.2× coverage per line. The unbiased genomic sampling provided by random short-read sequencing also allows insight into distributions of transposable elements and copy number polymorphisms found within populations and demonstrates that short-read sequencing methods provide an efficient means to quantify variation in genome organization and content. Continued development of methods for statistical inference of shallow-depth genome-wide sequencing data will allow such sparse, partial data sets to become the norm in the emerging field of population genomics.

Regional rates of recombination often correlate with levels of nucleotide diversity, and either selective or neutral hypotheses can explain this relationship. Regional recombination rates also correlate with nucleotide differences between human and chimpanzee, consistent with models where recombination is mutagenic; however, a lack of correlation is observed in the Drosophila melanogaster group, consistent with models invoking natural selection. Here, we revisit the relationship among recombination, diversity, and interspecies difference by generating empirical estimates of these parameters in Drosophila pseudoobscura . To measure recombination rate, we genotyped 1,294 backcross hybrids at 50 markers across the largest assembled linkage group in this species. Genome-wide diversity was estimated by sequencing a second isolate of D. pseudoobscura at shallow coverage. Alignment to the sequenced genome of the closely related species, Drosophila persimilis , provided nucleotide site orthology. Our findings demonstrate that scale is critical in determining correlates to recombination rate: fine-scale cross-over rate estimates are far stronger predictors of both diversity and interspecies difference than broad-scale estimates. The correlation of fine-scale recombination rate to diversity and interspecies difference appears to be genome-wide, evidenced by examination of an X-linked region in greater detail. Because we observe a strong correlation of cross-over rate with interspecies difference, even after correcting for segregating ancestral variation, we suggest that both mutagenic and selective forces generate these correlations, the latter in regions of low crossing over. We propose that it is not cross-overs per se that are mutagenic, but rather repair of DNA double-strand break precursors via crossing over and gene conversion.

Laryngeal cancer disproportionately affects more African-Americans than European-Americans. Here, we analyze the genome-wide somatic point mutations from the tumors of 13 African-Americans and 57 European-Americans from TCGA to differentiate between environmental and ancestrally-inherited factors. The mean number of mutations were different between African-Americans (151.31) and European-Americans (277.63). Other differences in the overall mutational landscape between African-American and European-American were also found. The frequency of C>A, and C>G were significantly different between the two populations (p-value

Charles Darwin remarked that "males, with their superior strength, pugnacity, armaments, unwieldly passion and love songs, are almost always the more active and most often, the initiators of sexual interactions". Here, we propose that such male sex drive directly impacts the genome by leading to its progressive masculinization--genes that possess sex-specific effects on male fitness accumulate to a much greater extent and are generally more diverged. The larger proportion of male versus female fitness modifiers in combination with stronger sexual selection may generate evolutionary signatures such as a greater sensitivity to male sterility and a paucity of X-linked male-specific genes. Male sex-drive theory complements the female-choice theory of sexual selection and allows for the genetic variation of costly sexual traits to be continuously replenished.

Much is known about the biology of Drosophila melanogaster. As a model organism, a comprehensive understanding of its development, physiology and reproduction has been acquired. As a result, a broad variety of transferable genetic tools and information has allowed sibling species of the D. melanogaster complex to emerge as an important speciation model system. By comparing D. melanogaster with its close relative, Drosophila simulans, as well as its other sibling species, we are beginning to understand the nature of genetic changes during the early stages of speciation. In general, we find that genes and traits involved in sex and reproduction are more variable. A large assortment of genes and traits that are involved in various aspects of mating and fertility reveal diagnostic differences between these sibling species. Sex and reproduction-related (SRR) genes are, on average, more diverged than genes with no apparent reproductive function. Furthermore, SRR genes appear more permissive at opting in novel function. These results follow a general trend observed in other taxa and demonstrate the preferential involvement of SRR genes in reproductive isolation and species formation.

While developmentally regulated genes are generally conserved, transformer (tra), a key locus involved in the regulation of sexual differentiation, is highly diverged between species of Drosophila. With an aim to understand its divergence between sibling species, we investigated tra sequence variation among members of the Drosophila melanogaster species complex, D. melanogaster, D. simulans, D. mauritiana, and D. sechellia. In this species group, tra divergence is rapid yet clocklike and exhibits large differences in protein size. D. melanogaster contains a 13-amino acid tandem duplication, whereas D. sechellia possesses a 72-amino acid tandem duplication representing a 30% increase in total amino acid residues. We also found evidence of a nonrandom distribution of replacement substitutions and heterogeneity in substitution rates using clustering statistics and a codon substitution model. We show that tra's rapid divergence in this species complex is the result of generally lower selective constraints around regions that encode arginine-serine (RS) domains and a significantly higher rate of substitutions around the insertion site of D. sechellia's large duplication. The proximity of rapidly diverged regions to sites of nucleotide insertion suggests that higher local rates of mutation may provide a causal mechanism for TRA's rapid divergence in this subgroup. A comparison of tra orthologs across the genus Drosophila suggest that TRA maintains an assortment of RS domains for proper sex determining function while much of the protein evolves relatively unconstrained.

It has been five years since Ernst Mayr, one of the most influential evolutionary biologists of the last century, passed away. Mayr’s seminal work as a naturalist and, in particular, as a bird systematist allowed him to approach the species problem in a revolutionary way. As a leading architect of the Modern Evolutionary Synthesis, Mayr helped integrate Darwinian theory with the broad fields of systematics and genetics. We pay tribute to this legend by publishing an interview taken shortly before his death.

The role of reinforcement in speciation can be explained by two distinct models. In model I, two diverged populations hybridize and produce fertile hybrids that successfully backcross (hybridization with gene flow). In model II, two populations hybridize but succeeding backcrosses are unproductive (hybridization without gene flow). Using Drosophila persimilis and D. pseudoobscura, we have tested model I by comparing the extent of heterospecific introgression in sympatric versus allopatric populations. We show that certain expectations of this particular model of reinforcement, which is based on hybridization and gene flow between divergent populations after secondary contact, are not realized in these two species. The evidence consists of the similarity of genetic distances as well as proportions of unique/rare alleles between sympatric and allopatric heterospecific populations and a negative correlation between genetic distance and geographical distance between heterospecific populations, which suggests ecological differentiation. This approach in quantifying differential gene flow has important consequences to studies that compare sympatric and allopatric isolation using genetic distance. Following model I, one would expect a pattern of higher prezygotic isolation in sympatric species compared to allopatric species of the same genetic distance simply as a result of an underestimation of genetic distance due to introgression between sympatric populations. We suggest more parsimonious explanations such as reinforcement without genetic exchange (model II) and ecological differentiation, which require high levels of preexisting reproductive isolation between populations.

In this paper, we review the literature on the growing body of data demonstrating the rapid evolution of sex and reproduction related (SRR) genes and show how a paradigm shift to the study of SRR genes can provide new approaches to solving some of the old problems in evolutionary biology. The argument is based on (1) the growing scope and importance of sexual selection in evolution, (2) the growing number of case studies showing rapid evolution of sexual traits in a wide variety of taxa, (3) the faster rate of DNA sequence divergence in genes affecting sexual function and fertility, (4) the evidence for the involvement of novel traits/genes in sexual functions, and (5) a proposed sex/non-sex dichotomy of the gene pool affecting viability versus fertility. It is argued that the adoption of the sex/non-sex dichotomy of genes/traits can provide new perspectives on such problems as species concepts, modes (allopatric/sympatric) of speciation, Haldane's rule, reinforcement, and the founder effect. It is proposed that the evolutionary study of genes affecting viability versus fertility is the key to understanding the genetic basis of speciation.

In accordance with Haldane's rule, hybridizations between species of the Drosophila simulans clade produce fertile females but sterile males. In this study, a comprehensive characterization was undertaken on the six types of F1 males that were the result of the crosses between D. simulans, D. sechellia, and D. mauritiana. With the use of light and electron microscopy, it was shown that while each particular hybrid genotype exhibited a specific sterility phenotype, these phenotypes fell into two distinct classes. The two hybrid genotypes that possessed D. mauritiana X-chromosomes contained spermatogenic defects that caused arrests in premeiotic spermatogenic stages. The other four F1 hybrids possessed postmeiotic spermatogenic defects. Nonsynchronous cell divisions, underdeveloped mitochondrial derivative-axonemal associations, and microtubule abnormalities were common to all of these hybrids. Each particular postmeiotically defective hybrid genotype demonstrated characteristically distinct profiles in sperm bundle number in addition to characteristic spermiogenic arrests in the furthest developed spermatids. These results in species hybrids contrast with the absence of significant differences in spermatogenic characters between species of this clade. In addition, by utilizing an attached-X cross, we investigated the influence of maternal effects and cytoplasmic factors on the sterility of D. simulans F1 hybrids and found none. However, we discovered a strain of D. simulans (2119) that caused a large shift in sterility from postmeiotic to premeiotic when crossed to D. sechellia. This suggests that D. simulans is polymorphic for genes involving premeiotic and postmeiotic sterility and that the two types of sterilities between species may have a simple genetic basis.

Background Comparative studies of amniotes have been hindered by a dearth of reptilian molecular sequences. With the genomic assembly of the green anole, Anolis carolinensis available, non-avian reptilian genes can now be compared to mammalian, avian, and amphibian homologs. Furthermore, with more than 350 extant species in the genus Anolis, anoles are an unparalleled example of tetrapod genetic diversity and divergence. As an important ecological, genetic and now genomic reference, it is imperative to develop a standardized Anolis gene nomenclature alongside associated vocabularies and other useful metrics. Results Here we report the formation of the Anolis Gene Nomenclature Committee (AGNC) and propose a standardized evolutionary characterization code that will help researchers to define gene orthology and paralogy with tetrapod homologs, provide a system for naming novel genes in Anolis and other reptiles, furnish abbreviations to facilitate comparative studies among the Anolis species and related iguanid squamates, and classify the geographical origins of Anolis subpopulations. Conclusions This report has been generated in close consultation with members of the Anolis and genomic research communities, and using public database resources including NCBI and Ensembl. Updates will continue to be regularly posted to new research community websites such as lizardbase. We anticipate that this standardized gene nomenclature will facilitate the accessibility of reptilian sequences for comparative studies among tetrapods and will further serve as a template for other communities in their sequencing and annotation initiatives., BMC Genomics, 12, ISSN:1471-2164

The second special issue on the evolution of sex-related traits and genes brings together a wide variety of papers that explore issues dealing with diverse topics such as behavior, organismal defense systems, molecular evolution, speciation, and genomics. This broad diversity reflects the recent expansion of the field of reproductive biology and evolution. The review by R. S. Singh and S. Jagadeeshan provides a historical perspective that describes the breadth of work that has emanated from early protein electrophoresis studies to the genomics-based approaches that are more common today. The authors illustrate how the study of sex- and reproductive-related (SRR) genes has now impacted fields of evolution spanning from selection and speciation to gene birth and evolutionary developmental genetics. A review article and a research article address questions related to mating preferences across two very different systems, Tetrahymena and Drosophila. S. S. Phadke et al. address an interesting question about the evolution of sex: does the evolution of more than one sex necessarily lead to the evolution of mating preferences? Using the ciliate species, Tetrahymena thermophila, which has up to seven “sexes,” they tested the mating frequencies of four of the sexes to determine if they exhibited mating preferences. Their results suggest that mating is random among the four sexes, thus concluding that the evolution of multiple sexes does not necessarily lead to the evolution of mate preferences. They discuss their findings in the context of ciliate evolution as well as the evolution of sex in general. A review by A. J. Moehring and M. Laturney offers a clear insight into our current understanding of the genetic basis and evolution of sexual isolation between species. The review focuses on female mate preference as a premating behavioural barrier and highlights two interesting commonalities across a wide variety of Drosophila species: different genes control conspecific and heterospecific male choice and a preferential location of genes for heterospecific male rejection in areas of low recombination. Another paper in this issue deals with the question of reproductive isolation between species. J. L. Marshall and N. DiRienzo address a central question in evolutionary biology about whether the same genetic and developmental pathways contribute to reproductive isolation at both intraspecific and interspecific levels. The authors characterize a postmating, prezygotic phenotype, the ability of males to induce egglaying in females, between diverging populations, and species of crickets. Using mating assays and RNAi, the authors demonstrate similar decreases in female fecundity and the abundance of a particular female protein within populations as well as between species. While their results are suggestive of a connection between incompatibilities found within species and reproductive isolation between species, the authors also discuss alternative explanations and the need for future work. Three papers address a topic not covered in our previous 2011 issue—the relationship between sex and organismal defense systems. In a review article focusing on whether or not different sexes evolve different defensive traits to avoid being harmed or eaten by a predator, G. Avila-Sakar and C. A. Romanow synthesize the literature on the hypothesis of male-biased herbivory in dioecious plant species. They outline shortcomings with the studies supporting the male-biased herbivory hypothesis and suggest a set of alternatives. Moreover, they outline a protocol that they suggest should be used to study plant-herbivore evolution in relation to this important question. Two research articles from L. C. Harshman's lab address questions linked to the evolution of the immune system in Drosophila. In one paper, J. Ma et al. compare males and females for measures of metabolic rate and locomotion in populations that have been selected for survival under an infection regime with Bacillus cereus. The authors characterize the potential physiological costs associated with mounting an effective and elevated immune response by measuring respiration rates (per fly or adjusted by weight), in addition to behavioural responses as measured by overall fly movement. The authors find evidence of a male-biased response with only the males responding metabolically to selection on elevated immunity. In their other contribution, the authors embark on a selection for survival to infection experiment and identify two pleiotropic responses (i.e., an increase in egg production and delayed development time). Of particular interest to this issue is the identified relationship between reproductive fitness in the form of egglaying and immunity. Three research articles use molecular biology and evolutionary genomics approaches to study diverse phenomena with respect to sex and evolution. R. L. Kanippayoor and A. J. Moehring address the functional and evolutionary consequences of the timing of protamine expression during the reorganization of the hereditary material in mature spermatozoa. Using transgenic Drosophila, they demonstrate that protamines are expressed from both alleles in diploid cells prior to meiosis, in contrast with the postmeiotic haploid expression of mammalian protamines. This work opens the door to future studies to ascertain the evolutionary benefits of diploid versus haploid expression of protamines, especially as it pertains to the fertilization success of sperm and an individual's fitness. By characterizing the evolution of the Izumo gene family in mammals, P. Grayson and A. Civetta explore how gene duplications can contribute to the adaptations on male reproductive traits. The Izumo gene family is comprised of four genes known to be expressed in sperm and possibly involved in sperm-egg interactions. The authors find contrasting patterns of molecular evolution which suggest a variety of evolutionary processes acting across different taxa. They conclude that such differing lineage-specific patterns of selection found in this gene family lend support to protein subfunctionalization as opposed to neofunctionalization or gene loss and represents species-specific adaptations to male fertility. In the final paper of this issue, to identify interlocus conflict in the genome, M. E. B. Hansen and R. J. Kulathinal generate and characterize male- and female-biased networks using the extensive genomic resources available in Drosophila. The authors integrate sex-specific expression data from modENCODE with known interaction data to identify putative direct and indirect interactions between male-biased genes and female-biased genes. Using this approach, the authors were able to demonstrate that a larger than expected fraction of the genome may potentially be involved in sexually antagonistic interactions at the molecular level. This special issue on the evolution of sex-related traits and genes contains a wonderfully diverse sample of studies that addresses both old and new questions in evolutionary reproductive biology. The editors would like to thank the dedicated and generous contributions of all authors and reviewers for helping to make this issue so interesting. Alberto Civetta Jose M. Eirin-Lopez Rob Kulathinal Jeremy L. Marshall

Background Genome-wide analysis of sequence divergence among species offers profound insights into the evolutionary processes that shape lineages. When full-genome sequencing is not feasible for a broad comparative study, we propose the use of array-based comparative genomic hybridization (aCGH) in order to identify orthologous genes with high sequence divergence. Here we discuss experimental design, statistical power, success rate, sources of variation and potential confounding factors. We used a spotted PCR product microarray platform from Drosophila melanogaster to assess sequence divergence on a gene-by-gene basis in three fully sequenced heterologous species (D. sechellia, D. simulans, and D. yakuba). Because complete genome assemblies are available for these species this study presents a powerful test for the use of aCGH as a tool to measure sequence divergence. Results We found a consistent and linear relationship between hybridization ratio and sequence divergence of the sample to the platform species. At higher levels of sequence divergence (< 92% sequence identity to D. melanogaster) ~84% of features had significantly less hybridization to the array in the heterologous species than the platform species, and thus could be identified as "diverged". At lower levels of divergence (≥ 97% identity), only 13% of genes were identified as diverged. While ~40% of the variation in hybridization ratio can be accounted for by variation in sequence identity of the heterologous sample relative to D. melanogaster, other individual characteristics of the DNA sequences, such as GC content, also contribute to variation in hybridization ratio, as does technical variation. Conclusions Here we demonstrate that aCGH can accurately be used as a proxy to estimate genome-wide divergence, thus providing an efficient way to evaluate how evolutionary processes and genomic architecture can shape species diversity in non-model systems. Given the increased number of species for which microarray platforms are available, comparative studies can be conducted for many interesting lineages in order to identify highly diverged genes that may be the target of natural selection.

In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F1 hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems., Author Summary The transformation of populations into distinct species depends on whether hybridization, recombination, and subsequent gene introgression can be suppressed between diverging species. We use partial genome sequences to reconstruct this evolutionary process in the Drosophila pseudoobscura species subgroup, which includes the hybridizing species pair D. pseudoobscura pseudoobscura and D. persimilis. Recent models suggest that chromosomal inversions can facilitate the persistence of hybridizing species because of their effects on recombination, whereby inverted regions would exhibit higher nucleotide divergence than non-inverted regions. Indeed, D. pseudoobscura-D. persimilis nucleotide divergence outside these inverted regions is lower than within or near inversions, resembling D. ps. pseudoobscura levels of within-species nucleotide diversity. We also observe that recombination suppression in F1 hybrids extends greater than 2 Mbp outside the inversion breakpoints. Furthermore, when genomic sequence of D. persimilis is compared to two sister subspecies—the hybridizing subspecies, D. ps. pseudoobscura, and a non-hybridizing control subspecies, D. ps. bogotana—autosomal divergence is lower in the former, demonstrating recent gene exchange. These lines of evidence support a speciation model in which the two hybridizing species persist despite the presence of recent genic introgression in collinear regions of the genome because of the reduced recombinational effects of the inversions that distinguish them.

Large clusters of coexpressed tissue-specific genes are abundant on chromosomes of diverse species. The genes coordinately misexpressed in diverse diseases are also found in similar clusters, suggesting that evolutionarily conserved mechanisms regulate expression of large multigenic regions both in normal development and in its pathological disruptions. Studies on individual loci suggest that silent clusters of coregulated genes are embedded in repressed chromatin domains, often localized to the nuclear periphery. To test this model at the genome-wide scale, we studied transcriptional regulation of large testis-specific gene clusters in somatic tissues of Drosophila . These gene clusters showed a drastic paucity of known expressed transgene insertions, indicating that they indeed are embedded in repressed chromatin. Bioinformatics analysis suggested the major role for the B-type lamin, LamDm o , in repression of large testis-specific gene clusters, showing that in somatic cells as many as three-quarters of these clusters interact with LamDm o . Ablation of LamDm o by using mutants and RNAi led to detachment of testis-specific clusters from nuclear envelope and to their selective transcriptional up-regulation in somatic cells, thus providing the first direct evidence for involvement of the B-type lamin in tissue-specific gene repression. Finally, we found that transcriptional activation of the lamina-bound testis-specific gene cluster in male germ line is coupled with its translocation away from the nuclear envelope. Our studies, which directly link nuclear architecture with coordinated regulation of tissue-specific genes, advance understanding of the mechanisms underlying both normal cell differentiation and developmental disorders caused by lesions in the B-type lamins and interacting proteins.

The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster.

Affiliations des auteurs : cf page 216 de l'article; International audience; Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

The Sdic gene cluster at the base of the X-chromosome is unique to the lineage of Drosophila melanogaster. The repeating unit in the cluster was formed from a duplication and fusion of the genes, AnnX and Cdic, which juxtaposed the 3′ untranslated region of AnnX to the third intron of Cdic. AnnX encodes Annexin 10 and Cdic encodes a cytoplasmic dynein intermediate chain. The 3′ untranslated region of AnnX contains two promoter elements, including a testis-specific element, and Cdic intron 3 contains a third promoter element; together these elements result in testis-specific transcription of Sdic. The Sdic protein features a novel amino terminus derived in part from Cdic intron 3 which contains motifs similar to those in axonemal dyneins. It has been demonstrated that the Sdic protein becomes incorporated into the tails of mature sperm. The evolution of the Sdic cluster required several deletions, at least one insertion, at least eleven nudeotide substitutions, and an estimated tenfold tandem duplication, all of which took place in the 1–3 million years since the divergence of D. melanogaster from D. simulans. Evidence for the ongoing evolution of Sdic including a recent selective sweep is found in the low levels of polymorphism across neighboring genes in the region, a large number of fixed amino acid replacements relative to fixed synonymous nucleotide substitutions, and a frequency spectrum of polymorphic nucleotides skewed toward rare variants. The analysis of polymorphism and divergence in the Sdic region, however, is complicated by the possible effects of background selection caused by deleterious new mutations, owing to the reduced amount of recombination in the region associated with its proximity to centromeric heterochromatin. We present the rapid evolution of this novel gene as a fascinating example of male-driven evolution incurred by recurrent selective sweeps.

In this commentary, Rob Kulathinal describes two papers from the Perrimon laboratory, each describing a new online resource that can assist geneticists with the design of their RNAi experiments. Hu et al.’s “UP-TORR: online tool for accurate and up-to-date annotation of RNAi reagents” and “FlyPrimerBank: An online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents” are published, respectively, in this month’s issue of GENETICS and G3: Genes|Genomes|Genetics.

We compare the functional spectrum of protein evolution in two separate animal lineages with respect to two hypotheses: (1) rates of divergence are distributed similarly among functional classes within both lineages, indicating that selective pressure on the proteome is largely independent of organismic-level biological requirements; and (2) rates of divergence are distributed differently among functional classes within each lineage, indicating species-specific selective regimes impact genome-wide substitutional patterns. Integrating comparative genome sequence with data from tissue-specific expressed-sequence-tag (EST) libraries and detailed database annotations, we find a functional genomic signature of rapid evolution and selective constraint shared between mammalian and nematode lineages despite their extensive morphological and ecological differences and distant common ancestry. In both phyla, we find evidence of accelerated evolution among components of molecular systems involved in coevolutionary change. In mammals, lineage-specific fast evolving genes include those involved in reproduction, immunity, and possibly, maternal–fetal conflict. Likelihood ratio tests provide evidence for positive selection in these rapidly evolving functional categories in mammals. In contrast, slowly evolving genes, in terms of amino acid or insertion/deletion (indel) change, in both phyla are involved in core molecular processes such as transcription, translation, and protein transport. Thus, strong purifying selection appears to act on the same core cellular processes in both mammalian and nematode lineages, whereas positive and/or relaxed selection acts on different biological processes in each lineage.

One of the principal goals of population genetics is to understand the processes by which genetic variation within species (polymorphism) becomes converted into genetic differences between species (divergence). In this transformation, selective neutrality, near neutrality, and positive selection may each play a role, differing from one gene to the next. Synonymous nucleotide sites are often used as a uniform standard of comparison across genes on the grounds that synonymous sites are subject to relatively weak selective constraints and so may, to a first approximation, be regarded as neutral. Synonymous sites are also interdigitated with nonsynonymous sites and so are affected equally by genomic context and demographic factors. Hence a comparison of levels of polymorphism and divergence between synonymous sites and amino acid replacement sites in a gene is potentially informative about the magnitude of selective forces associated with amino acid replacements. We have analyzed 56 genes in which polymorphism data from D. simulans are compared with divergence from a reference strain of D. melanogaster. The framework of the analysis is Bayesian and assumes that the distribution of selective effects (Malthusian fitnesses) is Gaussian with a mean that differs for each gene. In such a model, the average scaled selection intensity (γ =N e s) of amino acid replacements eligible to become polymorphic or fixed is −7.31, and the standard deviation of selective effects within each locus is 6.79 (assuming homoscedasticity across loci). For newly arising mutations of this type that occur in autosomal or X-linked genes, the average proportion of beneficial mutations is 19.7%. Among the amino acid polymorphisms in the sample, the expected average proportion of beneficial mutations is 47.7%, and among amino acid replacements that become fixed the average proportion of beneficial mutations is 94.3%. The average scaled selection intensity of fixed mutations is +5.1. The presence of positive selection is pervasive with the single exception of kl-5, a Y-linked fertility gene. We find no evidence that a significant fraction of fixed amino acid replacements is neutral or nearly neutral or that positive selection drives amino acid replacements at only a subset of the loci. These results are model dependent and we discuss possible modifications of the model that might allow more neutral and nearly neutral amino acid replacements to be fixed.

Much is known about the biology of Drosophila melanogaster. As a model organism, a comprehensive understanding of its development, physiology and reproduction has been acquired. As a result, a broad variety of transferable genetic tools and information has allowed sibling species of the D. melanogaster complex to emerge as an important speciation model system. By comparing D. melanogaster with its close relative, Drosophila simulans, as well as its other sibling species, we are beginning to understand the nature of genetic changes during the early stages of speciation. In general, we find that genes and traits involved in sex and reproduction are more variable. A large assortment of genes and traits that are involved in various aspects of mating and fertility reveal diagnostic differences between these sibling species. Sex and reproduction-related (SRR) genes are, on average, more diverged than genes with no apparent reproductive function. Furthermore, SRR genes appear more permissive at opting in novel function. These results follow a general trend observed in other taxa and demonstrate the preferential involvement of SRR genes in reproductive isolation and species formation.

Relatively little is known about the importance of amino acid interactions in protein and phenotypic evolution. Here we examine whether mutations that are pathogenic in Drosophila melanogaster become fixed via epistasis in other Dipteran genomes. Overall divergence at pathogenic amino acid sites is reduced. However, ∼10% of the substitutions at these sites carry the exact same pathogenic amino acid found in D. melanogaster mutants. Hence compensatory mutation(s) must have evolved. Surprisingly, the fraction 10% is not affected by phylogenetic distance. These results support a selection-driven process that allows compensated amino acid substitutions to become rapidly fixed in taxa with large populations.

Background Domesticated animals quickly evolve docile and submissive behaviors after isolation from their wild conspecifics. Model organisms reared for prolonged periods in the laboratory also exhibit similar shifts towards these domesticated behaviors. Yet whether this divergence is due to inadvertent selection in the lab or the fixation of deleterious mutations remains unknown. Results Here, we compare the genomes of lab-reared and wild-caught Drosophila melanogaster to understand the genetic basis of these recently endowed behaviors common to laboratory models. From reassembled genomes of common lab strains, we identify unique, derived variants not present in global populations (lab-specific SNPs). Decreased selective constraints across low frequency SNPs (unique to one or two lab strains) are different from patterns found in the wild and more similar to neutral expectations, suggesting an overall accumulation of deleterious mutations. However, high-frequency lab SNPs found in most or all lab strains reveal an enrichment of X-linked loci and neuro-sensory genes across large extended haplotypes. Among shared polymorphisms, we also find highly differentiated SNPs, in which the derived allele is higher in frequency in the wild (Fst*wild>lab), enriched for similar neurogenetic ontologies, indicative of relaxed selection on more active wild alleles in the lab. Conclusions Among random mutations that continuously accumulate in the laboratory, we detect common adaptive signatures in domesticated lab strains of fruit flies. Our results demonstrate that lab animals can quickly evolve domesticated behaviors via unconscious selection by humans early on a broad pool of disproportionately large neurogenetic targets followed by the fixation of accumulated deleterious mutations on functionally similar targets. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0580-1) contains supplementary material, which is available to authorized users.

Background Behavior, while complex and dynamic, is among the most diverse, derived, and rapidly evolving traits in animals. The highly labile nature of heritable behavioral change is observed in such evolutionary phenomena as the emergence of converged behaviors in domesticated animals, the rapid evolution of preferences, and the routine development of ethological isolation between diverging populations and species. In fact, it is believed that nervous system development and its potential to evolve a seemingly infinite array of behavioral innovations played a major role in the successful diversification of metazoans, including our own human lineage. However, unlike other rapidly evolving functional systems such as sperm-egg interactions and immune defense, the genetic basis of rapid behavioral change remains elusive. Presentation of the hypothesis Here we propose that the rapid divergence and widespread novelty of innate and adaptive behavior is primarily a function of its genomic architecture. Specifically, we hypothesize that the broad diversity of behavioral phenotypes present at micro- and macroevolutionary scales is promoted by a disproportionately large mutational target of neurogenic genes. We present evidence that these large neuro-behavioral targets are significant and ubiquitous in animal genomes and suggest that behavior’s novelty and rapid emergence are driven by a number of factors including more selection on a larger pool of variants, a greater role of phenotypic plasticity, and/or unique molecular features present in large genes. We briefly discuss the origins of these large neurogenic genes, as they relate to the remarkable diversity of metazoan behaviors, and highlight key consequences on both behavioral traits and neurogenic disease across, respectively, evolutionary and ontogenetic time scales. Testing the hypothesis Current approaches to studying the genetic mechanisms underlying rapid phenotypic change primarily focus on identifying signatures of Darwinian selection in protein-coding regions. In contrast, the large mutational target hypothesis places genomic architecture and a larger allelic pool at the forefront of rapid evolutionary change, particularly in genetic systems that are polygenic and regulatory in nature. Genomic data from brain and neural tissues in mammals as well as a preliminary survey of neurogenic genes from comparative genomic data support this hypothesis while rejecting both positive and relaxed selection on proteins or higher mutation rates. In mammals and invertebrates, neurogenic genes harbor larger protein-coding regions and possess a richer regulatory repertoire of miRNA targets and transcription factor binding sites. Overall, neurogenic genes cover a disproportionately large genomic fraction, providing a sizeable substrate for evolutionary, genetic, and molecular mechanisms to act upon. Readily available comparative and functional genomic data provide unexplored opportunities to test whether a distinct neurogenomic architecture can promote rapid behavioral change via several mechanisms unique to large genes, and which components of this large footprint are uniquely metazoan. Implications of the hypothesis The large mutational target hypothesis highlights the eminent roles of mutation and functional genomic architecture in generating rapid developmental and evolutionary change. It has broad implications on our understanding of the genetics of complex adaptive traits such as behavior by focusing on the importance of mutational input, from SNPs to alternative transcripts to transposable elements, on driving evolutionary rates of functional systems. Such functional divergence has important implications in promoting behavioral isolation across short- and long-term timescales. Due to genome-scaled polygenic adaptation, the large target effect also contributes to our inability to identify adapted behavioral candidate genes. The presence of large neurogenic genes, particularly in the mammalian brain and other neural tissues, further offers emerging insight into the etiology of neurodevelopmental and neurodegenerative diseases. The well-known correlation between neurological spectrum disorders in children and paternal age may simply be a direct result of aging fathers accumulating mutations across these large neurodevelopmental genes. The large mutational target hypothesis can also explain the rapid evolution of other functional systems covering a large genomic fraction such as male fertility and its preferential association with hybrid male sterility among closely related taxa. Overall, a focus on mutational potential may increase our power in understanding the genetic basis of complex phenotypes such as behavior while filling a general gap in understanding their evolution. Electronic supplementary material The online version of this article (doi:10.1186/s13062-016-0162-1) contains supplementary material, which is available to authorized users.