Inhibition protects acquired song segments during vocal learning in zebra finches Daniela Vallentin1,2,*, Georg Kosche1,2,*, Dina Lipkind3, Michael A. Long1,2,† Science 15 Jan 2016: Vol. 351, Issue 6270, pp. 267-271 DOI: 10.1126/science.aad3023 Vocal imitation involves incorporating instructive auditory information into relevant motor circuits through processes that are poorly understood. In zebra finches, we found that exposure to a tutor’s song drives spiking activity within premotor neurons in the juvenile, whereas inhibition suppresses such responses upon learning in adulthood. We measured inhibitory currents evoked by the tutor song throughout development while simultaneously quantifying each bird’s learning trajectory. Surprisingly, we found that the maturation of synaptic inhibition onto premotor neurons is correlated with learning but not age. We used synthetic tutoring to demonstrate that inhibition is selective for specific song elements that have already been learned and not those still in refinement. Our results suggest that structured inhibition plays a crucial role during song acquisition, enabling a piece-by-piece mastery of complex tasks. Oxytocin-dependent consolation behavior in rodents J. P. Burkett1,2,3,*, E. Andari1,2,3, Z. V. Johnson1,2,3, D. C. Curry2,3, F. B. M. de Waal2,3,4, L. J. Young1,2,3,5,* Science 22 Jan 2016: Vol. 351, Issue 6271, pp. 375-378 DOI: 10.1126/science.aac4785 Consolation behavior toward distressed others is common in humans and great apes, yet our ability to explore the biological mechanisms underlying this behavior is limited by its apparent absence in laboratory animals. Here, we provide empirical evidence that a rodent species, the highly social and monogamous prairie vole (Microtus ochrogaster), greatly increases partner-directed grooming toward familiar conspecifics (but not strangers) that have experienced an unobserved stressor, providing social buffering. Prairie voles also match the fear response, anxiety-related behaviors, and corticosterone increase of the stressed cagemate, suggesting an empathy mechanism. Exposure to the stressed cagemate increases activity in the anterior cingulate cortex, and oxytocin receptor antagonist infused into this region abolishes the partner-directed response, showing conserved neural mechanisms between prairie vole and human. Ancient Babylonian astronomers calculated Jupiter’s position from the area under a time-velocity graph Mathieu Ossendrijver* Science 29 Jan 2016: Vol. 351, Issue 6272, pp. 482-484 DOI: 10.1126/science.aad8085 The idea of computing a body’s displacement as an area in time-velocity space is usually traced back to 14th-century Europe. I show that in four ancient Babylonian cuneiform tablets, Jupiter’s displacement along the ecliptic is computed as the area of a trapezoidal figure obtained by drawing its daily displacement against time. This interpretation is prompted by a newly discovered tablet on which the same computation is presented in an equivalent arithmetical formulation. The tablets date from 350 to 50 BCE. The trapezoid procedures offer the first evidence for the use of geometrical methods in Babylonian mathematical astronomy, which was thus far viewed as operating exclusively with arithmetical concepts. Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction Yasuhiro Yamauchi1,*, Jonathan M. Riel1,*, Victor A. Ruthig1,*, Eglė A. Ortega1, Michael J. Mitchell2, Monika A. Ward1,† Science 29 Jan 2016: Vol. 351, Issue 6272, pp. 514-516 DOI: 10.1126/science.aad1795 The mammalian Y chromosome is considered a symbol of maleness, as it encodes a gene driving male sex determination, Sry, as well as a battery of other genes important for male reproduction. We previously demonstrated in the mouse that successful assisted reproduction can be achieved when the Y gene contribution is limited to only two genes, Sry and spermatogonial proliferation factor Eif2s3y. Here, we replaced Sry by transgenic activation of its downstream target Sox9, and Eif2s3y, by transgenic overexpression of its X chromosome–encoded homolog Eif2s3x. The resulting males with no Y chromosome genes produced haploid male gametes and sired offspring after assisted reproduction. Our findings support the existence of functional redundancy between the Y chromosome genes and their homologs encoded on other chromosomes. Nature Super-resolution imaging reveals distinct chromatin folding for different epigenetic states Alistair N. Boettiger, Bogdan Bintu, Jeffrey R. Moffitt, Siyuan Wang, Brian J. Beliveau, Geoffrey Fudenberg, Maxim Imakaev, Leonid A. Mirny, Chao-ting Wu & Xiaowei Zhuang Nature 529, 418–422 (21 January 2016) doi:10.1038/nature16496 Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories1, 2, 3, 4, 5. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms2, 3, 4, 6, 7, 8, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression9, 10, 11. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation. Ancient balancing selection at tan underlies female colour dimorphism in Drosophila erecta Amir Yassin, Héloïse Bastide, Henry Chung, Michel Veuille, Jean R. David & John E. Pool Nature Communications Published 18 Jan 2016 DOI: 10.1038/ncomms10400 Dimorphic traits are ubiquitous in nature, but the evolutionary factors leading to dimorphism are largely unclear. We investigate a potential case of sexual mimicry in Drosophila erecta, in which females show contrasting resemblance to males. We map the genetic basis of this sex-limited colour dimorphism to a region containing the gene tan. We find a striking signal of ancient balancing selection at the ‘male-specific enhancer’ of tan, with exceptionally high sequence divergence between light and dark alleles, suggesting that this dimorphism has been adaptively maintained for millions of years. Using transgenic reporter assays, we confirm that these enhancer alleles encode expression differences that are predicted to generate this pigmentation dimorphism. These results are compatible with the theoretical prediction that divergent phenotypes maintained by selection can evolve simple genetic architectures. PLoS Evolutionary Novelty in a Butterfly Wing Pattern through Enhancer Shuffling Richard W. R. Wallbank, Simon W. Baxter, Carolina Pardo-Diaz, Joseph J. Hanly, Simon H. Martin, James Mallet, Kanchon K. Dasmahapatra, Camilo Salazar, Mathieu Joron, Nicola Nadeau, W. Owen McMillan, Chris D. Jiggins PLOS Published: January 15, 2016 DOI: 10.1371/journal.pbio.1002353 An important goal in evolutionary biology is to understand the genetic changes underlying novel morphological structures. We investigated the origins of a complex wing pattern found among Amazonian Heliconius butterflies. Genome sequence data from 142 individuals across 17 species identified narrow regions associated with two distinct red colour pattern elements, dennis and ray. We hypothesise that these modules in non-coding sequence represent distinct cis-regulatory loci that control expression of the transcription factor optix, which in turn controls red pattern variation across Heliconius. Phylogenetic analysis of the two elements demonstrated that they have distinct evolutionary histories and that novel adaptive morphological variation was created by shuffling these cis-regulatory modules through recombination between divergent lineages. In addition, recombination of modules into different combinations within species further contributes to diversity. Analysis of the timing of diversification in these two regions supports the hypothesis of introgression moving regulatory modules between species, rather than shared ancestral variation. The dennis phenotype introgressed into Heliconius melpomene at about the same time that ray originated in this group, while ray introgressed back into H. elevatus much more recently. We show that shuffling of existing enhancer elements both within and between species provides a mechanism for rapid diversification and generation of novel morphological combinations during adaptive radiation. Near-Stasis in the Long-Term Diversification of Mesozoic Tetrapods Roger B. J. Benson, Richard J. Butler, John Alroy, Philip D. Mannion, Matthew T. Carrano, Graeme T. Lloyd PLOS Published: January 25, 2016 DOI: 10.1371/journal.pbio.1002359 How did evolution generate the extraordinary diversity of vertebrates on land? Zero species are known prior to ~380 million years ago, and more than 30,000 are present today. An expansionist model suggests this was achieved by large and unbounded increases, leading to substantially greater diversity in the present than at any time in the geological past. This model contrasts starkly with empirical support for constrained diversification in marine animals, suggesting different macroevolutionary processes on land and in the sea. We quantify patterns of vertebrate standing diversity on land during the Mesozoic–early Paleogene interval, applying sample-standardization to a global fossil dataset containing 27,260 occurrences of 4,898 non-marine tetrapod species. Our results show a highly stable pattern of Mesozoic tetrapod diversity at regional and local levels, underpinned by a weakly positive, but near-zero, long-term net diversification rate over 190 million years. Species diversity of non-flying terrestrial tetrapods less than doubled over this interval, despite the origins of exceptionally diverse extant groups within mammals, squamates, amphibians, and dinosaurs. Therefore, although speciose groups of modern tetrapods have Mesozoic origins, rates of Mesozoic diversification inferred from the fossil record are slow compared to those inferred from molecular phylogenies. If high speciation rates did occur in the Mesozoic, then they seem to have been balanced by extinctions among older clades. An apparent 4-fold expansion of species richness after the Cretaceous/Paleogene (K/Pg) boundary deserves further examination in light of potential taxonomic biases, but is consistent with the hypothesis that global environmental disturbances such as mass extinction events can rapidly adjust limits to diversity by restructuring ecosystems, and suggests that the gradualistic evolutionary diversification of tetrapods was punctuated by brief but dramatic episodes of radiation. Genome-Wide Analysis Reveals Novel Regulators of Growth in Drosophila melanogaster Sibylle Chantal Vonesch, David Lamparter, Trudy F. C. Mackay, Sven Bergmann, Ernst Hafen PLOS Genetics DOI:10.1371/journal.pgen.1005616 January 11, 2016 Organismal size depends on the interplay between genetic and environmental factors. Genome-wide association (GWA) analyses in humans have implied many genes in the control of height but suffer from the inability to control the environment. Genetic analyses in Drosophila have identified conserved signaling pathways controlling size; however, how these pathways control phenotypic diversity is unclear. We performed GWA of size traits using the Drosophila Genetic Reference Panel of inbred, sequenced lines. We find that the top associated variants differ between traits and sexes; do not map to canonical growth pathway genes, but can be linked to these by epistasis analysis; and are enriched for genes and putative enhancers. Performing GWA on well-studied developmental traits under controlled conditions expands our understanding of developmental processes underlying phenotypic diversity. Fast turnover of genome transcription across evolutionary time exposes entire non-coding DNA to de novo gene emergence Rafik Neme, Diethard Tautz eLife DOI: http://dx.doi.org/10.7554/eLife.09977Published February 2, 2016 Deep sequencing analyses have shown that a large fraction of genomes is transcribed, but the significance of this transcription is much debated. Here, we characterize the phylogenetic turnover of poly-adenylated transcripts in a comprehensive sampling of taxa of the mouse (genus Mus), spanning a phylogenetic distance of 10 Myr. Using deep RNA sequencing we find that at a given sequencing depth transcriptome coverage becomes saturated within a taxon, but keeps extending when compared between taxa, even at this very shallow phylogenetic level. Our data show a high turnover of transcriptional states between taxa and that no major transcript-free islands exist across evolutionary time. This suggests that the entire genome can be transcribed into poly-adenylated RNA when viewed at an evolutionary time scale. We conclude that any part of the non-coding genome can potentially become subject to evolutionary functionalization via de novo gene evolution within relatively short evolutionary time spans. P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila Eric D Hoopfer, Yonil Jung, Hidehiko K Inagaki, Gerald M Rubin, David J Anderson eLife DOI: http://dx.doi.org/10.7554/eLife.11346 Published December 29, 2015 How brains are hardwired to produce aggressive behavior, and how aggression circuits are related to those that mediate courtship, is not well understood. A large-scale screen for aggression-promoting neurons in Drosophila identified several independent hits that enhanced both inter-male aggression and courtship. Genetic intersections revealed that 8-10 P1 interneurons, previously thought to exclusively control male courtship, were sufficient to promote fighting. Optogenetic experiments indicated that P1 activation could promote aggression at a threshold below that required for wing extension. P1 activation in the absence of wing extension triggered persistent aggression via an internal state that could endure for minutes. High-frequency P1 activation promoted wing extension and suppressed aggression during photostimulation, whereas aggression resumed and wing extension was inhibited following photostimulation offset. Thus, P1 neuron activation promotes a latent, internal state that facilitates aggression and courtship, and controls the overt expression of these social behaviors in a threshold-dependent, inverse manner. Modeling evolutionary transitions in social insects Michael Doebeli Ehab Abouheif eLife, Jan 2016 DOI: 10.7554/eLife.12721 Mathematical models based on direct fitness calculations may be able to explain important aspects of social evolution in insects. Bottlenecks and selective sweeps during domestication have increased deleterious genetic variation in dogs Clare D. Marsden, Diego Ortega-Del Vecchyo, Dennis P. O’Brien, Jeremy F. Taylor, Oscar Ramirez, Carles Vilà, Tomas Marques-Bonet, Robert D. Schnabel, Robert K. Wayne, and Kirk E. Lohmueller PNAS 2016 113 (1) 152-157; published ahead of print December 22, 2015, doi:10.1073/pnas.1512501113 Population bottlenecks, inbreeding, and artificial selection can all, in principle, influence levels of deleterious genetic variation. However, the relative importance of each of these effects on genome-wide patterns of deleterious variation remains controversial. Domestic and wild canids offer a powerful system to address the role of these factors in influencing deleterious variation because their history is dominated by known bottlenecks and intense artificial selection. Here, we assess genome-wide patterns of deleterious variation in 90 whole-genome sequences from breed dogs, village dogs, and gray wolves. We find that the ratio of amino acid changing heterozygosity to silent heterozygosity is higher in dogs than in wolves and, on average, dogs have 2–3% higher genetic load than gray wolves. Multiple lines of evidence indicate this pattern is driven by less efficient natural selection due to bottlenecks associated with domestication and breed formation, rather than recent inbreeding. Further, we find regions of the genome implicated in selective sweeps are enriched for amino acid changing variants and Mendelian disease genes. To our knowledge, these results provide the first quantitative estimates of the increased burden of deleterious variants directly associated with domestication and have important implications for selective breeding programs and the conservation of rare and endangered species. Specifically, they highlight the costs associated with selective breeding and question the practice favoring the breeding of individuals that best fit breed standards. Our results also suggest that maintaining a large population size, rather than just avoiding inbreeding, is a critical factor for preventing the accumulation of deleterious variants. Limitations of GCTA as a solution to the missing heritability problem Siddharth Krishna Kumar, Marcus W. Feldman, David H. Rehkopf, and Shripad Tuljapurkar PNAS 2016 113 (1) E61-E70; published ahead of print December 22, 2015, doi:10.1073/pnas.1520109113 Genome-wide association studies (GWASs) seek to understand the relationship between complex phenotype(s) (e.g., height) and up to millions of single-nucleotide polymorphisms (SNPs). Early analyses of GWASs are commonly believed to have “missed” much of the additive genetic variance estimated from correlations between relatives. A more recent method, genome-wide complex trait analysis (GCTA), obtains much higher estimates of heritability using a model of random SNP effects correlated between genotypically similar individuals. GCTA has now been applied to many phenotypes from schizophrenia to scholastic achievement. However, recent studies question GCTA’s estimates of heritability. Here, we show that GCTA applied to current SNP data cannot produce reliable or stable estimates of heritability. We show first that GCTA depends sensitively on all singular values of a high-dimensional genetic relatedness matrix (GRM). When the assumptions in GCTA are satisfied exactly, we show that the heritability estimates produced by GCTA will be biased and the standard errors will likely be inaccurate. When the population is stratified, we find that GRMs typically have highly skewed singular values, and we prove that the many small singular values cannot be estimated reliably. Hence, GWAS data are necessarily overfit by GCTA which, as a result, produces high estimates of heritability. We also show that GCTA’s heritability estimates are sensitive to the chosen sample and to measurement errors in the phenotype. We illustrate our results using the Framingham dataset. Our analysis suggests that results obtained using GCTA, and the results’ qualitative interpretations, should be interpreted with great caution. A ribosome-inactivating protein in a Drosophila defensive symbiont Phineas T. Hamiltona,1, Fangni Pengb, Martin J. Boulangerb, and Steve J. Perlmana,c,1 PNAS 2016 113 (2) 350-355; published ahead of print December 28, 2015, doi:10.1073/pnas.1518648113 Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense. Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake Ryan K. Schott, Johannes Müller, Clement G. Y. Yang, Nihar Bhattacharyya, Natalie Chan, Mengshu Xu, James M. Morrow, Ana-Hermina Ghenu, Ellis R. Loew, Vincent Tropepe, and Belinda S. W. Chang PNAS 2016 113 (2) 356-361; published ahead of print December 29, 2015, doi:10.1073/pnas.1513284113 Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the “transmutation” theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single “cones.” Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality. Evolution of maternal effect senescence Jacob A. Moorad and Daniel H. Nussey PNAS 2016 113 (2) 362-367; published ahead of print December 29, 2015, doi:10.1073/pnas.1520494113 Increased maternal age at reproduction is often associated with decreased offspring performance in numerous species of plants and animals (including humans). Current evolutionary theory considers such maternal effect senescence as part of a unified process of reproductive senescence, which is under identical age-specific selective pressures to fertility. We offer a novel theoretical perspective by combining William Hamilton’s evolutionary model for aging with a quantitative genetic model of indirect genetic effects. We demonstrate that fertility and maternal effect senescence are likely to experience different patterns of age-specific selection and thus can evolve to take divergent forms. Applied to neonatal survival, we find that selection for maternal effects is the product of age-specific fertility and Hamilton’s age-specific force of selection for fertility. Population genetic models show that senescence for these maternal effects can evolve in the absence of reproductive or actuarial senescence; this implies that maternal effect aging is a fundamentally distinct demographic manifestation of the evolution of aging. However, brief periods of increasingly beneficial maternal effects can evolve when fertility increases with age faster than cumulative survival declines. This is most likely to occur early in life. Our integration of theory provides a general framework with which to model, measure, and compare the evolutionary determinants of the social manifestations of aging. Extension of our maternal effects model to other ecological and social contexts could provide important insights into the drivers of the astonishing diversity of lifespans and aging patterns observed among species. Chromosome position determines the success of double-strand break repair Cheng-Sheng Lee, Ruoxi W. Wang, Hsiao-Han Chang, Daniel Capurso, Mark R. Segal, and James E. Haber PNAS 2016 113 (2) E146-E154; published ahead of print December 29, 2015, doi:10.1073/pnas.1523660113 Repair of a chromosomal double-strand break (DSB) by gene conversion depends on the ability of the broken ends to encounter a donor sequence. To understand how chromosomal location of a target sequence affects DSB repair, we took advantage of genome-wide Hi-C analysis of yeast chromosomes to create a series of strains in which an induced site-specific DSB in budding yeast is repaired by a 2-kb donor sequence inserted at different locations. The efficiency of repair, measured by cell viability or competition between each donor and a reference site, showed a strong correlation (r = 0.85 and 0.79) with the contact frequencies of each donor with the DSB repair site. Repair efficiency depends on the distance between donor and recipient rather than any intrinsic limitation of a particular donor site. These results further demonstrate that the search for homology is the rate-limiting step in DSB repair and suggest that cells often fail to repair a DSB because they cannot locate a donor before other, apparently lethal, processes arise. The repair efficiency of a donor locus can be improved by four factors: slower 5′ to 3′ resection of the DSB ends, increased abundance of replication protein factor A (RPA), longer shared homology, or presence of a recombination enhancer element adjacent to a donor. A rigorous comparison of sexual selection indexes via simulations of diverse mating systems Jonathan M. Henshaw, Andrew T. Kahn, and Karoline Fritzsche PNAS 2016 113 (3) E300-E308; published ahead of print January 6, 2016, doi:10.1073/pnas.1518067113 Sexual selection is a cornerstone of evolutionary theory, but measuring it has proved surprisingly difficult and controversial. Various proxy measures—e.g., the Bateman gradient and the opportunity for sexual selection—are widely used in empirical studies. However, we do not know how reliably these measures predict the strength of sexual selection across natural systems, and most perform poorly in theoretical worst-case scenarios. Here we provide a rigorous comparison of eight commonly used indexes of sexual selection. We simulated 500 biologically plausible mating systems, based on the templates of five well-studied species that cover a diverse range of reproductive life histories. We compared putative indexes to the actual strength of premating sexual selection, measured as the strength of selection on a simulated “mating trait.” This method sidesteps a key weakness of empirical studies, which lack an appropriate yardstick against which proxy measures can be assessed. Our model predicts that, far from being useless, the best proxy measures reliably track the strength of sexual selection across biologically realistic scenarios. The maximum intensity of precopulatory sexual selection s′maxs′max (the Jones index) outperformed all other indexes and was highly correlated with the strength of sexual selection. In contrast, the Bateman gradient and the opportunity for sexual selection were poor predictors of sexual selection, despite their continuing popularity. Sex-specific silencing of X-linked genes by Xist RNA Srimonta Gayen, Emily Maclary, Michael Hinten, and Sundeep Kalantry PNAS 2016 113 (3) E309-E318; published ahead of print January 6, 2016, doi:10.1073/pnas.1515971113 X-inactive specific transcript (Xist) long noncoding RNA (lncRNA) is thought to catalyze silencing of X-linked genes in cis during X-chromosome inactivation, which equalizes X-linked gene dosage between male and female mammals. To test the impact of Xist RNA on X-linked gene silencing, we ectopically induced endogenous Xist by ablating the antisense repressor Tsix in mice. We find that ectopic Xist RNA induction and subsequent X-linked gene silencing is sex specific in embryos and in differentiating embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). A higher frequency of XΔTsixY male cells displayed ectopic Xist RNA coating compared with XΔTsixX female cells. This increase reflected the inability of XΔTsixY cells to efficiently silence X-linked genes compared with XΔTsixX cells, despite equivalent Xist RNA induction and coating. Silencing of genes on both Xs resulted in significantly reduced proliferation and increased cell death in XΔTsixX female cells relative to XΔTsixY male cells. Thus, whereas Xist RNA can inactivate the X chromosome in females it may not do so in males. We further found comparable silencing in differentiating XΔTsixY and 39,XΔTsix (XΔTsixO) ESCs, excluding the Y chromosome and instead implicating the X-chromosome dose as the source of the sex-specific differences. Because XΔTsixX female embryonic epiblast cells and EpiSCs harbor an inactivated X chromosome prior to ectopic inactivation of the active XΔTsix X chromosome, we propose that the increased expression of one or more X-inactivation escapees activates Xist and, separately, helps trigger X-linked gene silencing. Decanalization of wing development accompanied the evolution of large wings in high-altitude Drosophila Justin B. Lack, Matthew J. Monette, Evan J. Johanning, Quentin D. Sprengelmeyer, and John E. Pool PNAS 2016 113 (4) 1014-1019; published ahead of print January 11, 2016, doi:10.1073/pnas.1515964113 In higher organisms, the phenotypic impacts of potentially harmful or beneficial mutations are often modulated by complex developmental networks. Stabilizing selection may favor the evolution of developmental canalization—that is, robustness despite perturbation—to insulate development against environmental and genetic variability. In contrast, directional selection acts to alter the developmental process, possibly undermining the molecular mechanisms that buffer a trait’s development, but this scenario has not been shown in nature. Here, we examined the developmental consequences of size increase in highland Ethiopian Drosophila melanogaster. Ethiopian inbred strains exhibited much higher frequencies of wing abnormalities than lowland populations, consistent with an elevated susceptibility to the genetic perturbation of inbreeding. We then used mutagenesis to test whether Ethiopian wing development is, indeed, decanalized. Ethiopian strains were far more susceptible to this genetic disruption of development, yielding 26 times more novel wing abnormalities than lowland strains in F2 males. Wing size and developmental perturbability cosegregated in the offspring of between-population crosses, suggesting that genes conferring size differences had undermined developmental buffering mechanisms. Our findings represent the first observation, to our knowledge, of morphological evolution associated with decanalization in the same tissue, underscoring the sensitivity of development to adaptive change. MBE Parallel evolution of copy-number variation across continents in Drosophila melanogaster Daniel R. Schrider, Matthew W. Hahn and David J. Begun ref Genetic differentiation across populations that is maintained in the presence of gene flow is a hallmark of spatially varying selection. In Drosophila melanogaster, the latitudinal clines across the eastern coasts of Australia and North America appear to be examples of this type of selection, with recent studies showing that a substantial portion of the D. melanogaster genome exhibits allele frequency differentiation with respect to latitude on both continents. As of yet there has been no genome-wide examination of differentiated copy-number variants (CNVs) in these geographic regions, despite their potential importance for phenotypic variation in Drosophila and other taxa. Here, we present an analysis of geographic variation in CNVs in D. melanogaster. We also present the first genomic analysis of geographic variation for copy-number variation in the sister species, D. simulans, in order to investigate patterns of parallel evolution in these close relatives. In D. melanogaster we find hundreds of CNVs, many of which show parallel patterns of geographic variation on both continents, lending support to the idea that they are influenced by spatially varying selection. These findings support the idea that polymorphic CNVs contribute to local adaptation in D. melanogaster. In contrast, we find very few CNVs in D. simulans that are geographically differentiated in parallel on both continents, consistent with earlier work suggesting that clinal patterns are weaker in this species. Evolution of reduced post-copulatory molecular interactions in Drosophila populations lacking sperm competition B. Hollis, D. Houle and T. J. Kawecki1 DOI: 10.1111/jeb.12763 In many species with internal fertilization, molecules transferred in the male ejaculate trigger and interact with physiological changes in females. It is controversial to what extent these interactions between the sexes act synergistically to mediate the female switch to a reproductive state or instead reflect sexual antagonism evolved as a by product of sexual selection on males. To address this question, we eliminated sexual selection by enforcing monogamy in populations of Drosophila melanogaster for 65 generations and then measured the expression of male seminal fluid protein genes and genes involved in the female response to mating. In the absence of sperm competition, male and female reproductive interests are perfectly aligned and any antagonism should be reduced by natural selection. Consistent with this idea, males from monogamous populations showed reduced expression of seminal fluid protein genes, 16% less on average than in polygamous males. Further, we identified 428 genes that responded to mating in females. After mating, females with an evolutionary history of monogamy exhibited lower relative expression of genes that were up regulated in response to mating and higher expression of genes that were down-regulated – in other words, their post-mating transcriptome appeared more virgin-like. Surprisingly, these genes showed a similar pattern even before mating, suggesting that monogamous females evolved to be less poised for mating and the accompanying receipt of male seminal fluid proteins. This reduced investment by both monogamous males and females in molecules involved in post-copulatory interactions points to a pervasive role of sexual conflict in shaping these interactions. Ancient Male Recombination Shaped Genetic Diversity of Neo-Y Chromosome in Drosophila albomicans Kazuhiro Satomura and Koichiro Tamura Mol Biol Evol (2016) 33 (2): 367-374. doi: 10.1093/molbev/msv221 Researchers studying Y chromosome evolution have drawn attention to neo-Y chromosomes in Drosophila species due to their resembling the initial stage of Y chromosome evolution. In the studies of neo-Y chromosome of Drosophila miranda, the extremely low genetic diversity observed suggested various modes of natural selection acting on the nonrecombining genome. However, alternative possibility may come from its peculiar origin from a single chromosomal fusion event with male achiasmy, which potentially caused and maintained the low genetic diversity of the neo-Y chromosome. Here, we report a real case where a neo-Y chromosome is in transition from an autosome to a typical Y chromosome. The neo-Y chromosome of Drosophila albomicans harbored a rich genetic diversity comparable to its gametologous neo-X chromosome and an autosome in the same genome. Analyzing sequence variations in 53 genes and measuring recombination rates between pairs of loci by cross experiments, we elucidated the evolutionary scenario of the neo-Y chromosome of D. albomicans having high genetic diversity without assuming selective force, i.e., it originated from a single chromosomal fusion event, experienced meiotic recombination during the initial stage of evolution and diverged from neo-X chromosome by the suppression of recombination tens or a few hundreds of thousand years ago. Consequently, the observed high genetic diversity on the neo-Y chromosome suggested a strong effect of meiotic recombination to introduce genetic variations into the newly arisen sex chromosome. Purifying selection on exonic splice enhancers in intronless genes Rosina Savisaar and Laurence D. Hurst, MBE Advance Access published January 23, 2016 Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few-exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. The Gut Microbiota Modulates Energy Metabolism in the Hibernating Brown Bear Ursus arctos Felix Sommer, Marcus Ståhlman, Olga Ilkayeva, Jon M. Arnemo, Jonas Kindberg, Johan Josefsson, Christopher B. Newgard, Ole Fröbert, Fredrik Bäckhed Cell Feb 2016 DOI: http://dx.doi.org/10.1016/j.celrep.2016.01.026 Hibernation is an adaptation that helps many animals to conserve energy during food shortage in winter. Brown bears double their fat depots during summer and use these stored lipids during hibernation. Although bears seasonally become obese, they remain metabolically healthy. We analyzed the microbiota of free-ranging brown bears during their active phase and hibernation. Compared to the active phase, hibernation microbiota had reduced diversity, reduced levels of Firmicutes and Actinobacteria, and increased levels of Bacteroidetes. Several metabolites involved in lipid metabolism, including triglycerides, cholesterol, and bile acids, were also affected by hibernation. Transplantation of the bear microbiota from summer and winter to germ-free mice transferred some of the seasonal metabolic features and demonstrated that the summer microbiota promoted adiposity without impairing glucose tolerance, suggesting that seasonal variation in the microbiota may contribute to host energy metabolism in the hibernating brown bear. Data-Driven Reversible Jump for QTL Mapping Daiane Aparecida Zuanetti, Luis Aparecido Milan Genetics January 5, 2016 vol. 202 no. 1 25-36; DOI: 10.1534/genetics.115.180802 We propose a birth–death–merge data-driven reversible jump (DDRJ) for multiple-QTL mapping where the phenotypic trait is modeled as a linear function of the additive and dominance effects of the unknown QTL genotypes. We compare the performance of the proposed methodology, usual reversible jump (RJ) and multiple-interval mapping (MIM), using simulated and real data sets. Compared with RJ, DDRJ shows a better performance to estimate the number of QTLs and their locations on the genome mainly when the QTLs effect is moderate, basically as a result of better mixing for transdimensional moves. The inclusion of a merge step of consecutive QTLs in DDRJ is efficient, under tested conditions, to avoid the split of true QTL’s effects between false QTLs and, consequently, selection of the wrong model. DDRJ is also more precise to estimate the QTLs location than MIM in which the number of QTLs need to be specified in advance. As DDRJ is more efficient to identify and characterize QTLs with smaller effect, this method also appears to be useful and brings contributions to identifying single-nucleotide polymorphisms (SNPs) that usually have a small effect on phenotype. The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila Yuriy A. Abramov, Aleksei S. Shatskikh, Oksana G. Maksimenko, Silvia Bonaccorsi, Vladimir A. Gvozdev, Sergey A. Lavrov Genetics January 5, 2016 vol. 202 no. 1 93-106; DOI: 10.1534/genetics.115.181693 Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo–formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2. Genome-wide SNPs reveal fine-scale differentiation among wingless alpine stonefly populations and introgression between winged and wingless forms Nicolas Dussex1, Aaron Chuah2 andJonathan M. Waters1 Evolution Article first published online: 10 DEC 2015 DOI: 10.1111/evo.12826 Volume 70, Issue 1, pages 38–47, January 2016 Insect flight loss is a repeated phenomenon in alpine habitats, where wing reduction is thought to enhance local recruitment and increase fecundity. One predicted consequence of flight loss is reduced dispersal ability, which should lead to population genetic differentiation and perhaps ultimately to speciation. Using a dataset of 15,123 SNP loci, we present comparative analyses of fine-scale population structure in codistributed Zelandoperla stonefly species, across three parallel altitudinal transects in New Zealand's Rock and Pillar mountain range. We find that winged populations (altitude 200–500 m; Zelandoperla decorata) show no genetic structuring within or among streams, suggesting substantial dispersal mediated by flight. By contrast, wingless populations (Zelandoperla fenestrata; altitude 200–1100 m) exhibit distinct genetic clusters associated with each stream, and additional evidence of isolation by distance within streams. Our data support the hypothesis that wing-loss can initiate diversification in alpine insect populations over small spatial scales. The often deep phylogenetic placement of lowland Z. fenestrata within their stream-specific clades suggests the possibility of independent alpine colonization events for each stream. Additionally, the detection of winged, interspecific hybrid individuals raises the intriguing possibility that a previously flightless lineage could reacquire flight via introgression. Morpho morphometrics: Shared ancestry and selection drive the evolution of wing size and shape in Morpho butterflies Nicolas Chazot, Stephen Panara, Nicolas Zilbermann, Patrick Blandin, Yann Le Poul, Raphaël Cornette, Marianne Elias† andVincent Debat† Evolution Article first published online: 7 JAN 2016 DOI: 10.1111/evo.12842 Volume 70, Issue 1, pages 181–194, January 2016 Butterfly wings harbor highly diverse phenotypes and are involved in many functions. Wing size and shape result from interactions between adaptive processes, phylogenetic history, and developmental constraints, which are complex to disentangle. Here, we focus on the genus Morpho (Nymphalidae: Satyrinae, 30 species), which presents a high diversity of sizes, shapes, and color patterns. First, we generate a comprehensive molecular phylogeny of these 30 species. Next, using 911 collection specimens, we quantify the variation of wing size and shape across species, to assess the importance of shared ancestry, microhabitat use, and sexual selection in the evolution of the wings. While accounting for phylogenetic and allometric effects, we detect a significant difference in wing shape but not size among microhabitats. Fore and hindwings covary at the individual and species levels, and the covariation differs among microhabitats. However, the microhabitat structure in covariation disappears when phylogenetic relationships are taken into account. Our results demonstrate that microhabitat has driven wing shape evolution, although it has not strongly affected forewing and hindwing integration. We also found that sexual dimorphism of forewing shape and color pattern are coupled, suggesting a common selective force. The genetic basis of color-related local adaptation in a ring-like colonization around the Mediterranean Reto Burri, Sylvain Antoniazza, Arnaud Gaigher, Anne-Lyse Ducrest, Céline Simon, The European Barn Owl Network, Luca Fumagalli, Jérôme Goudet, and Alexandre Roulin Evolution Article first published online: 15 DEC 2015 DOI: 10.1111/evo.12824 Volume 70, Issue 1, pages 140–153, January 2016 Uncovering the genetic basis of phenotypic variation and the population history under which it established is key to understand the trajectories along which local adaptation evolves. Here, we investigated the genetic basis and evolutionary history of a clinal plumage color polymorphism in European barn owls (Tyto alba). Our results suggest that barn owls colonized the Western Palearctic in a ring-like manner around the Mediterranean and meet in secondary contact in Greece. Rufous coloration appears to be linked to a recently evolved nonsynonymous-derived variant of the melanocortin 1 receptor (MC1R) gene, which according to quantitative genetic analyses evolved under local adaptation during or following the colonization of Central Europe. Admixture patterns and linkage disequilibrium between the neutral genetic background and color found exclusively within the secondary contact zone suggest limited introgression at secondary contact. These results from a system reminiscent of ring species provide a striking example of how local adaptation can evolve from derived genetic variation. CRISPR Cas9 Rationally engineered Cas9 nucleases with improved specificity Ian M. Slaymaker, Linyi Gao, Bernd Zetsche, David A. Scott, Winston X. Yan, Feng Zhang Science 01 Jan 2016: Vol. 351, Issue 6268, pp. 84-88 DOI: 10.1126/science.aad5227 The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects Benjamin P. Kleinstiver, Vikram Pattanayak, Michelle S. Prew, Shengdar Q. Tsai, Nhu T. Nguyen, Zongli Zheng & J. Keith Joung Nature 529, 490–495 (28 January 2016) doi:10.1038/nature16526 CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases. The Anthropocene is functionally and stratigraphically distinct from the Holocene Colin N. Waters1,*, Jan Zalasiewicz2, Colin Summerhayes3, Anthony D. Barnosky4, Clément Poirier5, Agnieszka Gałuszka6, Alejandro Cearreta7, Matt Edgeworth8, Erle C. Ellis9, Michael Ellis1, Catherine Jeandel10, Reinhold Leinfelder11, J. R. McNeill12, Daniel deB. Richter13, Will Steffen14, James Syvitski15, Davor Vidas16, Michael Wagreich17, Mark Williams2, An Zhisheng18, Jacques Grinevald19, Eric Odada20, Naomi Oreskes21, Alexander P. Wolfe22 Science Science 08 Jan 2016: Vol. 351, Issue 6269, pp. DOI: 10.1126/science.aad2622 Human activity is leaving a pervasive and persistent signature on Earth. Vigorous debate continues about whether this warrants recognition as a new geologic time unit known as the Anthropocene. We review anthropogenic markers of functional changes in the Earth system through the stratigraphic record. The appearance of manufactured materials in sediments, including aluminum, plastics, and concrete, coincides with global spikes in fallout radionuclides and particulates from fossil fuel combustion. Carbon, nitrogen, and phosphorus cycles have been substantially modified over the past century. Rates of sea-level rise and the extent of human perturbation of the climate system exceed Late Holocene changes. Biotic changes include species invasions worldwide and accelerating rates of extinction. These combined signals render the Anthropocene stratigraphically distinct from the Holocene and earlier epochs. Reviews Genetics of Adaptation Hopi Hoekstra Annual Review of Genetics Vol. 50 (Volume publication date December 2016) Theodosius Dobzhansky on Hybrid Sterility and Speciation Jerry A. Coyne Genetics January 5, 2016 vol. 202 no. 1 5-7; DOI: 10.1534/genetics.115.184770 Sewall Wright on Evolution in Mendelian Populations and the “Shifting Balance” Nicholas H. Barton Genetics January 5, 2016 vol. 202 no. 1 3-4; DOI: 10.1534/genetics.115.184796 The Soft Touch: Low-Affinity Transcription Factor Binding Sites in Development and Evolution Justin Crocker, Ella Preger-Ben Noon, David L. Stern Current Topics in Developmental Biology ISSN 0070-2153 http://dx.doi.org/10.1016/bs.ctdb.2015.11.018 Transcription factor proteins regulate gene expression by binding to specific DNA regions. Most studies of transcription factor binding sites have focused on the highest affinity sites for each factor. There is abundant evidence, however, that binding sites with a range of affinities, including very low affinities, are critical to gene regulation. Here, we present the theoretical and experimental evidence for the importance of low-affinity sites in gene regulation and development. We also discuss the implications of the widespread use of low-affinity sites in eukaryotic genomes for robustness, precision, specificity, and evolution of gene regulation. Other Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites David L. Stern bioRxiv preprint first posted online Jan. 22, 2016; doi: http://dx.doi.org/10.1101/037762 . Multiple methods have been introduced over the past 30 years to identify the genomic insertion sites of transposable elements and other DNA elements that integrate into genomes. However, each of these methods suffer from limitations that can frustrate attempts to map multiple insertions in a single genome and to map insertions in genomes of high complexity that contain extensive repetitive DNA. I introduce a new method for transposon mapping that is simple to perform, can accurately map multiple insertions per genome, and generates long sequence “reads” that facilitate mapping to complex genomes. The method, called TagMap, for Tagmentation-based Mapping, relies on a modified Tn5 tagmentation protocol with a single tagmentation adaptor followed by PCR using primers specific to the tranposable element and the adaptor sequence. Several minor modifications to normal tagmentation reagents and protocols allow easy and rapid preparation of TagMap libraries. Short read sequencing starting from the adaptor sequence generates oriented reads that flank and are oriented toward the transposable element insertion site. The convergent orientation of adjacent reads at the insertion site allows straightforward prediction of the precise insertion site(s). A Linux shell script is provided to identify insertion sites from fastq files. Species-specific differences in the behavior of male tsetse fly genitalia hidden in the female during copulation R.D. B RICEÑO, W.G. E BERHARD, E. CHINEA-CANO, D. W EGRZYNEK and T. DOS SANTOS ROLO Ethology Ecology & Evolution, 2016 Vol. 28, No. 1, 53–76, http://dx.doi.org/10.1080/03949370.2014.1002114 A long-standing question in morphological evolution is why male genitalia tend to diverge more rapidly than other structures. One possible explanation is that male genitalia are under sexual selection to function as ‘internal courtship devices’. Males of closely related species may provide divergent stimulation using different genital morphologies and behaviors. Testing this hypothesis has been difficult, however, because the presumed genital courtship behavior is often hidden from view inside the female, and because studies of how the male’s genitalia interact with those of the female are nearly always limited to a single species in a given group, thus restricting opportunities for comparison of closely related species. We present new morphological and behavioral data for portions of the male genitalia that are hidden in the female during copulation in five species in the tsetse fly genus Glossina using data from dissections of pairs frozen in copula, artificially stimulated males, and from copulating pairs viewed with a new X-ray technique that allows events inside the female to be recorded in real time. These data almost certainly give only an incomplete view of this complex, previously hidden world. But even so they clearly reveal that, as predicted by sexual selection theory, the male genitalia of Glossina flies perform dramatic, stereotyped, rhythmic movements deep within the female’s reproductive tract and in inward folds of her external surface, and that many of these movements probably differ among closely related species. Most of the movements are not explicable as means by which the male anchors himself more securely to the female; all are likely to result in stimulation of the female. A female Glossina can be stimulated tactilely at a given moment during copulation at up to 8–10 or more different sites on her body.