Biology of the Cell Biology of the Cell DOI: 10.1111/boc.201400094 Left-right asymmetry in the light of TOR: An update on what we know so far Teresa Casar Tena, Martin D. Burkhalter and Melanie Philipp The internal left-right (LR) asymmetry is a characteristic that exists throughout the animal kingdom from roundworms over flies and fish to mammals. Cilia, which are antenna-like structures protruding into the extracellular space, are involved in establishing LR asymmetry during early development. Humans who suffer from dysfunctional cilia often develop conditions such as heterotaxy, where internal organs appear to be placed randomly. As a consequence to this failure in asymmetry development, serious complications such as congenital heart defects (CHD) occur. The mammalian (or mechanistic) target of rapamycin (mTOR) pathway has recently emerged as an important regulator regarding symmetry breaking. The mTOR pathway governs fundamental processes such as protein translation or metabolism. Its activity can be transduced by two complexes, which are called TORC1 and TORC2, respectively. So far, only TORC1 has been implicated with asymmetry development and appears to require very precise regulation. A number of recent papers provided evidence that dysregulated TORC1 results in alterations of motile cilia and asymmetry defects. In here, we give an update on what we know so far of mTORC1 in LR asymmetry development. Bio Web of Conferences BIO Web of Conferences 4, 00005 (2015) DOI: 10.1051/bioconf/20150400005 Tracing the origin of our species through palaeogenomics Eva-Maria Geigl, E. Andrew Bennett, and Thierry Grange Abstract. The recent breathtaking progress in whole genome sequencing technology allows access to the genomes both of ancient organisms and populations, including those now extinct. Despite the heavy degradation and the extremely low quantities of ancient DNA, it is sometimes possible to sequence an entire genome from a fossil. This enterprise has been successful in the case of fossilized remains from Neanderthals, a lineage of hominids that lived in Europe for 200,000 years and disappeared 30,000 years ago. An even greater surprise was the genome that has been obtained from a small finger bone preserved in a cave in the Siberian Altai Mountains. This genome revealed the existence of a human lineage previously unknown from the fossil record. The corresponding population mixed with the Neanderthals and the ancestors of the present day populations of South-East Asia. These hybridization events left different traces in the non-African human populations emphasizing the fact that we are genomic mosaics. The comparison of the different genomes also gives hints to how the genome of present-day populations was shaped and helps us to better understand which parts of our genetic make-up are responsible for the biological features of H. sapiens. Current Biology Current Biology 25, 1619–1624, June 15, 2015 Flies Remember the Time of Day Nitin S. Chouhan, 1 Reinhard Wolf, 1 Charlotte Helfrich-Fo ̈rster, 2 and Martin Heisenberg 1, * The circadian clock enables organisms to anticipate daily environmental cycles and drives corresponding changes in behavior [1, 2]. Such endogenous oscillators also enable animals to display time-specific memory [1, 3–5]. For instance, mice and honeybees associate the location of a stimulus (like food or mate) with a certain time of day (time-place learning) [6, 7]. However, the mechanism underlying time-related learning and memory is not known. In the present study, we investigate time-specific odor learning. We use a genetically tractable animal, the fly Drosophila melanogaster. Starved flies are trained in the morning and afternoon to associate distinct odors with sucrose reward. The training is repeated the next day, and their time-dependent odor preference is tested on the third day. Our results indicate that Drosophila can express appetitive memory at the relevant time of day if the two conditioning events are separated by more than 4 hr. Flies can form time-odor associations in constant darkness (DD) as well as in a daily light-dark (LD) cycle, but not when kept under constant light (LL) conditions. Circadian clock mutants, period 01 (per 01 ) and clock AR (clk AR ), learned to associate sucrose reward with a certain odor but were unable to form time-odor associations. Our findings show that flies can utilize temporal information as an additional cue in appetitive learning. Time-odor learning in flies depends on a per- and clk-dependent endogenous mechanism that is independent of environmental light cues. Development Development 142, 1971-1977. doi: 10.1242/dev.119404 Diversity of epithelial morphogenesis during eggshell formation in drosophilids Miriam Osterfield, Trudi Schüpbach, Eric Wieschaus and Stanislav Y. Shvartsman The eggshells of drosophilid species provide a powerful model for studying the origins of morphological diversity. The dorsal appendages, or respiratory filaments, of these eggshells display a remarkable interspecies variation in number and shape, and the epithelial patterning underlying the formation of these structures is an area of active research. To extend the analysis of dorsal appendage formation to include morphogenesis, we developed an improved 3D image reconstruction approach. This approach revealed considerable interspecies variation in the cell shape changes and neighbor exchanges underlying appendage formation. Specifically, although the appendage floor in Drosophila melanogaster is formed through spatially ordered neighbor exchanges, the same structure in Scaptodrosophila pattersoni is formed through extreme changes in cell shape, whereas Drosophila funebris appears to display a combination of both cellular mechanisms. Furthermore, localization patterns of Par3/Bazooka suggest a self-organized, cell polarity-based origin for the variability of appendage number in S. pattersoni. Our results suggest that species deploy different combinations of apically and basally driven mechanisms to convert a two-dimensional primordium into a three-dimensional structure, and provide new directions for exploring the molecular origins of interspecies morphological variation. Developmental Cell Developmental Cell, Volume 33, Issue 6, p675–689, 22 June 2015 DOI: http://dx.doi.org/10.1016/j.devcel.2015.04.026 The Atypical Cadherin Dachsous Controls Left-Right Asymmetry in Drosophila Nicanor González-Morales, Charles Géminard, Gaëlle Lebreton, Delphine Cerezo, Jean-Baptiste Coutelis, Stéphane Noselli Left-right (LR) asymmetry is essential for organ development and function in metazoans, but how initial LR cue is relayed to tissues still remains unclear. Here, we propose a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis. We further show that the Ds/Fat and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non-autonomous propagation of early LR asymmetry. eLife eLife 2015;4:e06203. DOI: 10.7554/eLife.06203 Female mice ultrasonically interact with males during courtship displays Joshua P Neunuebel, Adam L Taylor, Ben J Arthur, SE Roian Egnor During courtship males attract females with elaborate behaviors. In mice, these displays include ultrasonic vocalizations. Ultrasonic courtship vocalizations were previously attributed to the courting male, despite evidence that both sexes produce virtually indistinguishable vocalizations. Because of this similarity, and the difficulty of assigning vocalizations to individuals, the vocal contribution of each individual during courtship is unknown. To address this question, we developed a microphone array system to localize vocalizations from socially interacting, individual adult mice. With this system, we show that female mice vocally interact with males during courtship. Males and females jointly increased their vocalization rates during chases. Furthermore, a female’s participation in these vocal interactions may function as a signal that indicates a state of increased receptivity. Our results reveal a novel form of vocal communication during mouse courtship, and lay the groundwork for a mechanistic dissection of communication during social behavior. Evolution Evolution, 10 JUN 2015, DOI: 10.1111/evo.12687 Commentary: When does understanding phenotypic evolution require identification of the underlying genes? Mark D. Rausher and Lynda F. Delph Adaptive evolution is fundamentally a genetic process. Over the past three decades, characterizing the genes underlying adaptive phenotypic change has revealed many important aspects of evolutionary change. At the same time, natural selection is often fundamentally an ecological process that can often be studied without identifying the genes underlying the variation on which it acts. This duality has given rise to disagreement about whether, and under what circumstances, it is necessary to identify specific genes associated with phenotypic change. This issue is of practical concern, especially for researchers who study non-model organisms, because of the often enormous cost and labor required to ―go for the genes‖. We here consider a number of situations and questions commonly addressed by researchers. Our conclusion is that while gene identification can be crucial for answering some questions, there are others for which definitive answers can be obtained without finding underlying genes. It should thus not be assumed that considerations of ―empirical completeness‖ dictate that gene identification is always desirable Evolution, DOI: 10.1111/evo.12685 No evidence for external genital morphology affecting cryptic female choice and reproductive isolation in Drosophila Hélène LeVasseur-Viens, Michal Polak and Amanda J. Moehring Genitalia are one of the most rapidly diverging morphological features in animals. The evolution of genital morphology is proposed to be driven by sexual selection via cryptic female choice, whereby a female selectively uptakes and uses a particular male's sperm on the basis of male genital morphology. The resulting shifts in genital morphology within a species can lead to divergence in genitalia between species, and consequently to reproductive isolation and speciation. Although this conceptual framework is supported by correlative data, there is little direct empirical evidence. Here, we used a microdissection laser to alter the morphology of the external male genitalia in Drosophila, a widely used genetic model for both genital shape and cryptic female choice. We evaluate the effect of precision alterations to lobe morphology on both interspecific and intraspecific mating, and demonstrate experimentally that the male genital lobes do not affect copulation duration or cryptic female choice, contrary to long-standing assumptions regarding the role of the lobes in this model system. Rather, we demonstrate that the lobes are essential for copulation to occur. Moreover, slight alterations to the lobes significantly reduced copulatory success only in competitive environments, identifying precopulatory sexual selection as a potential contributing force behind genital diversification. Genetics Genetics: Early Online, published on June 19, 2015 as 10.1534/genetics.115.178491 Genetics of intra-species variation in avoidance behavior induced by a thermal stimulus in C. elegans Rajarshi Ghosh, Joshua S. Bloom, Aylia Mohammadi, Molly E. Schumer, Peter Andolfatto, William Ryu, Leonid Kruglyak Individuals within a species vary in their responses to a wide range of stimuli, partly as a result of differences in their genetic makeup. Relatively little is known about the genetic and neuronal mechanisms contributing to diversity of behavior in natural populations. By studying intra-species variation in innate avoidance behavior to thermal stimuli in the nematode Caenorhabditis elegans, we uncovered genetic principles of how different components of a behavioral response can be altered in nature to generate behavioral diversity. Using a thermal pulse assay, we uncovered heritable variation in responses to a transient temperature increase. Quantitative trait locus mapping revealed that separate components of this response were controlled by distinct genomic loci. The loci we identified contributed to variation in components of thermal pulse avoidance behavior in an additive fashion. Our results show that the escape behavior induced by thermal stimuli is composed of simpler behavioral components that are influenced by at least six distinct genetic loci. The loci that decouple components of the escape behavior reveal a genetic system that allows independent modification of behavioral parameters. Our work sets the foundation for future studies of evolution of innate behaviors at the molecular and neuronal level. Mol. Biol. Evol. Mol. Biol. Evol. 32(6):1567–1580 doi:10.1093/molbev/msv048 Digestive Organ in the Female Reproductive Tract Borrows Genes from Multiple Organ Systems to Adopt Critical Functions Camille Meslin, Melissa S. Plakke, Aaron B. Deutsch, Brandon S. Small, Nathan I. Morehouse, and Nathan L. Clark Persistent adaptive challenges are often met with the evolution of novel physiological traits. Although there are specific examples of single genes providing new physiological functions, studies on the origin of complex organ functions are lacking. One such derived set of complex functions is found in the Lepidopteran bursa copulatrix, an organ within the female reproductive tract that digests nutrients from the male ejaculate or spermatophore. Here, we characterized bursa physiology and the evolutionary mechanisms by which it was equipped with digestive and absorptive functionality. By studying the transcriptome of the bursa and eight other tissues, we revealed a suite of highly expressed and secreted gene products providing the bursa with a combination of stomach-like traits for mechanical and enzymatic digestion of the male spermatophore. By subsequently placing these bursa genes in an evolutionary framework, we found that the vast majority of their novel digestive functions were co-opted by borrowing genes that continue to be expressed in non-reproductive tissues. However, a number of bursa-specific genes have also arisen, some of which represent unique gene families restricted to Lepidoptera and may provide novel bursa-specific functions. This pattern of promiscuous gene borrowing and relatively infrequent evolution of tissue-specific duplicates stands in contrast to studies of the evolution of novelty via single gene co-option. Our results suggest that the evolution of complex organ-level phenotypes may often be enabled (and subsequently constrained) by changes in tissue specificity that allow expression of existing genes in novel contexts, such as reproduction. The extent to which the selective pressures encountered in these novel roles require resolution via duplication and sub/neofunctionalization is likely to be determined by the need for specialized reproductive functionality. Thus, complex physiological phenotypes such as that found in the bursa offer important opportunities for understanding the relative role of pleiotropy and specialization in adaptive evolution. Molecular Phylogenetics and Evolution Molecular Phylogenetics and Evolution Volume 92, November 2015, Pages 226–242, doi:10.1016/j.ympev.2015.06.014 Rapid adaptive radiation and host plant conservation in the Hawaiian picture wing Drosophila (Diptera: Drosophilidae) ☆ Karl N. Magnacca, Donald K. Price The Hawaiian picture wing Drosophila are a striking example of adaptive radiation in specialist saprophages on an island system. We use DNA sequences from five nuclear genes with a total of 4260 nucleotides to provide a comprehensive phylogeny and biogeographic analysis of 90 species in the Hawaiian Drosophila picture wing clade. The current analysis indicates that the evolution of the picture wing clade took place more recently than previously suggested. The relationships of several morphologically anomalous taxa are resolved with strong support. Biogeography and host plant analyses show two periods of rapid divergence occurred when Kauai and Oahu were the main high islands, indicating that a combination of complex topographical features of islands and development of novel host plant associations was key to the rapid diversification of these lineages. For the past 2 million years, host associations within lineages have been largely stable, and speciation has occurred primarily due to the establishment of populations on newer islands as they arose followed by divergence by isolation. The existence of several apparently relictual taxa suggests that extinction has also played a major role in assembly of the present Hawaiian Drosophila fauna. Nature Nature 522, 81–84 (04 June 2015) doi:10.1038/nature14249 Ancient proteins resolve the evolutionary history of Darwin’s South American ungulates Frido Welker, Matthew J. Collins, Jessica A. Thomas, Marc Wadsley, Selina Brace, Enrico Cappellini, Samuel T. Turvey, Marcelo Reguero, Javier N. Gelfo, Alejandro Kramarz, Joachim Burger, Jane Thomas-Oates, David A. Ashford, Peter D. Ashton, Keri Rowsell, Duncan M. Porter, Benedikt Kessler, Roman Fischer, Carsten Baessmann, Stephanie Kaspar, Jesper V. Olsen, Patrick Kiley, James A. Elliott, Christian D. Kelstrup, Victoria Mullin et al. No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as ‘South American native ungulates’. To Charles Darwin1, 2, who first collected their remains, they included perhaps the ‘strangest animal[s] ever discovered’. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago3, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria4, 5, 6. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies7, 8, 9. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed5, 6, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates4, 6 from ‘condylarths’, a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time. Nature 522, 167–172 (11 June 2015) doi:10.1038/nature14507 Population genomics of Bronze Age Eurasia Morten E. Allentoft, Martin Sikora, Karl-Göran Sjögren, Simon Rasmussen, Morten Rasmussen, Jesper Stenderup, Peter B. Damgaard, Hannes Schroeder, Torbjörn Ahlström, Lasse Vinner, Anna-Sapfo Malaspinas, Ashot Margaryan, Tom Higham, David Chivall, Niels Lynnerup, Lise Harvig, Justyna Baron, Philippe Della Casa, Paweł Dąbrowski, Paul R. Duffy, Alexander V. Ebel, Andrey Epimakhov, Karin Frei, Mirosław Furmanek, Tomasz Gralak et al. The Bronze Age of Eurasia (around 3000–1000 BC) was a period of major cultural changes. However, there is debate about whether these changes resulted from the circulation of ideas or from human migrations, potentially also facilitating the spread of languages and certain phenotypic traits. We investigated this by using new, improved methods to sequence low-coverage genomes from 101 ancient humans from across Eurasia. We show that the Bronze Age was a highly dynamic period involving large-scale population migrations and replacements, responsible for shaping major parts of present-day demographic structure in both Europe and Asia. Our findings are consistent with the hypothesized spread of Indo-European languages during the Early Bronze Age. We also demonstrate that light skin pigmentation in Europeans was already present at high frequency in the Bronze Age, but not lactose tolerance, indicating a more recent onset of positive selection on lactose tolerance than previously thought. Nature 522, 470–473 (25 June 2015) doi:10.1038/nature14419 Sexual selection protects against extinction Alyson J. Lumley, Łukasz Michalczyk, James J. N. Kitson, Lewis G. Spurgin, Catriona A. Morrison, Joanne L. Godwin, Matthew E. Dickinson, Oliver Y. Martin, Brent C. Emerson, Tracey Chapman & Matthew J. G. Gage Reproduction through sex carries substantial costs, mainly because only half of sexual adults produce offspring1. It has been theorized that these costs could be countered if sex allows sexual selection to clear the universal fitness constraint of mutation load2, 3, 4. Under sexual selection, competition between (usually) males and mate choice by (usually) females create important intraspecific filters for reproductive success, so that only a subset of males gains paternity. If reproductive success under sexual selection is dependent on individual condition, which is contingent to mutation load, then sexually selected filtering through ‘genic capture’5 could offset the costs of sex because it provides genetic benefits to populations. Here we test this theory experimentally by comparing whether populations with histories of strong versus weak sexual selection purge mutation load and resist extinction differently. After evolving replicate populations of the flour beetle Tribolium castaneum for 6 to 7 years under conditions that differed solely in the strengths of sexual selection, we revealed mutation load using inbreeding. Lineages from populations that had previously experienced strong sexual selection were resilient to extinction and maintained fitness under inbreeding, with some families continuing to survive after 20 generations of sib × sib mating. By contrast, lineages derived from populations that experienced weak or non-existent sexual selection showed rapid fitness declines under inbreeding, and all were extinct after generation 10. Multiple mutations across the genome with individually small effects can be difficult to clear, yet sum to a significant fitness load; our findings reveal that sexual selection reduces this load, improving population viability in the face of genetic stress. Nature Reviews Genetics Nature Reviews Genetics 16, 321–332 (2015) doi:10.1038/nrg3920 Machine learning applications in genetics and genomics Maxwell W. Libbrecht & William Stafford Noble The field of machine learning, which aims to develop computer algorithms that improve with experience, holds promise to enable computers to assist humans in the analysis of large, complex data sets. Here, we provide an overview of machine learning applications for the analysis of genome sequencing data sets, including the annotation of sequence elements and epigenetic, proteomic or metabolomic data. We present considerations and recurrent challenges in the application of supervised, semi-supervised and unsupervised machine learning methods, as well as of generative and discriminative modelling approaches. We provide general guidelines to assist in the selection of these machine learning methods and their practical application for the analysis of genetic and genomic data sets. Nature Reviews Genetics 16, 333–343 (2015) doi:10.1038/nrg3931 Estimating the mutation load in human genomes Brenna M. Henn, Laura R. Botigué, Carlos D. Bustamante, Andrew G. Clark & Simon Gravel Next-generation sequencing technology has facilitated the discovery of millions of genetic variants in human genomes. A sizeable fraction of these variants are predicted to be deleterious. Here, we review the pattern of deleterious alleles as ascertained in genome sequencing data sets and ask whether human populations differ in their predicted burden of deleterious alleles — a phenomenon known as mutation load. We discuss three demographic models that are predicted to affect mutation load and relate these models to the evidence (or the lack thereof) for variation in the efficacy of purifying selection in diverse human genomes. We also emphasize why accurate estimation of mutation load depends on assumptions regarding the distribution of dominance and selection coefficients — quantities that remain poorly characterized for current genomic data sets. Nature Reviews Genetics 16, 409–420 (2015) doi:10.1038/nrg3950 Determinants of the rate of protein sequence evolution Jianzhi Zhang & Jian-Rong Yang The rate and mechanism of protein sequence evolution have been central questions in evolutionary biology since the 1960s. Although the rate of protein sequence evolution depends primarily on the level of functional constraint, exactly what determines functional constraint has remained unclear. The increasing availability of genomic data has enabled much needed empirical examinations on the nature of functional constraint. These studies found that the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance. A combination of theoretical and empirical analyses has identified multiple mechanisms behind these observations and demonstrated a prominent role in protein evolution of selection against errors in molecular and cellular processes. Nature Reviews Genetics 16, 379–394 (2015) doi:10.1038/nrg3927 Methods for the directed evolution of proteins Michael S. Packer & David R. Liu Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties. This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins. We highlight recent cases in which directed evolution generated enzymatic activities and substrate specificities not known to exist in nature. PLoS Genetics PLOS Genetics | DOI:10.1371/journal.pgen.1005279 Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species Winslow C. Johnson, Alison J. Ordway, Masayoshi Watada, Jonathan N. Pruitt, Thomas M. Williams, Mark Rebeiz The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pig- ment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits. silencer element mutations cause intraspecific pigmentation differences ... PNAS PNAS | May 26, 2015 | vol. 112 | no. 21 | 6659–6663 www.pnas.org/cgi/doi/10.1073/pnas.1500758112 The recent invasion of natural Drosophila simulans populations by the P-element Robert Kofler, Tom Hill, Viola Nolte, Andrea J. Betancourt, and Christian Schlötterer The P-element is one of the best understood eukaryotic transposable elements. It invaded Drosophila melanogaster populations within a few decades but was thought to be absent from close rel atives, including Drosophila simulans. Five decades after the spread in D. melanogaster, we provide evidence that the P-element has also invaded D. simulans. P-elements in D. simulans appear to have been acquired recently from D. melanogaster probably via a single horizontal transfer event. Expression data indicate that the P-element is processed in the germ line of D. simulans, and genomic data show an enrichment of P-element insertions in putative origins of replication, similar to that seen in D. melanogaster. This ongoing spread of the P-element in natural populations provides a unique opportunity to understand the dynamics of transposable element spread and the associated piwi-interacting RNAs defense mechanisms. PNAS 2015 112 (26) 8036-8040; published ahead of print June 15, 2015, doi:10.1073/pnas.1501844112 Mx1 and Mx2 key antiviral proteins are surprisingly lost in toothed whales Benjamin A. Braun, Amir Marcovitz, J. Gray Camp, Robin Jia, and Gill Bejerano Everybody loves dolphins. And orcas. And Mx (Myxovirus) genes. Mx genes are important immune genes that help mammals fight many RNA and DNA viruses, including HIV, measles, and flu. We make a surprising discovery: dolphins, orcas, and likely all toothed whales lost both Mx genes soon after they diverged from baleen whales and ungulates, which preserve these important genes intact. Because both genes were likely lost simultaneously, we speculate that a viral outbreak exploiting the Mx genes may have forced the toothed whale’s ancestor to sacrifice both. Because the Mx genes are so important, and because all 56 nontoothed whale sequenced mammals carry Mx genes, our discovery makes an important contribution to help preserve these magnificent mammals. Viral outbreaks in dolphins and other Delphinoidea family members warrant investigation into the integrity of the cetacean immune system. The dynamin-like GTPase genes Myxovirus 1 (Mx1) and Mx2 defend mammals against a broad range of viral infections. Loss of Mx1 function in human and mice enhances infectivity by multiple RNA and DNA viruses, including orthomyxoviruses (influenza A), paramyxoviruses (measles), and hepadnaviruses (hepatitis B), whereas loss of Mx2 function leads to decreased resistance to HIV-1 and other viruses. Here we show that both Mx1 and Mx2 have been rendered nonfunctional in Odontoceti cetaceans (toothed whales, including dolphins and orcas). We discovered multiple exon deletions, frameshift mutations, premature stop codons, and transcriptional evidence of decay in the coding sequence of both Mx1 and Mx2 in four species of Odontocetes. We trace the likely loss event for both proteins to soon after the divergence of Odontocetes and Mystocetes (baleen whales) ∼33–37 Mya. Our data raise intriguing questions as to what drove the loss of both Mx1 and Mx2 genes in the Odontoceti lineage, a double loss seen in none of 56 other mammalian genomes, and suggests a hitherto unappreciated fundamental genetic difference in the way these magnificent mammals respond to viral infections. PNAS 2015 112 (27) 8168-8169; published ahead of print June 29, 2015, doi:10.1073/pnas.1510121112 Therese Ann Markow Drosophila reproduction: Molecules meet morphology Our understanding of how male seminal fluid proteins (SFPs) influence the outcome of copulation in a wide range of insect taxa has been fueled primarily by studies of Drosophila (1). SFPs are known to localize, after copulation, to specific regions of the female reproductive tract and nervous system. Manipulative studies revealed that these proteins control a number of postcopulatory processes, such as sperm storage and retrieval and female egg production and remating (2). Proteins produced by the female reproductive tract have been identified (3, 4) and also mediate molecular interactions between the sexes. The influences of SFPs on female reproduction have been considered largely in a purely physiological way. A newly characterized and critical aspect of male–female interactions is revealed in the PNAS paper by Mattei et al. (5), who used micro-CT scans to prepare 3D reconstructions of the female reproductive tract before, during, and after mating. Copulation produces a dynamic series of dramatic conformational changes in the mated female reproductive tract, changes never visible previously in studies with dissected material. Moreover, Mattei et al. (5) demonstrate the roles of particular male seminal components in mediating these morphological changes inside the female. In the age of molecular biology and signaling, interest in more traditional descriptive disciplines, such as morphology, became obsolete. Now, however, we see that morphology is anything but static and that, in fact, the coordinated changes in morphology following mating are what are ultimately responsible for whether reproduction will be completed. PNAS, July 7, 2015, vol. 112 no. 27, 8475–8480, doi: 10.1073/pnas.1505797112 Integrated 3D view of postmating responses by the Drosophila melanogaster female reproductive tract, obtained by micro-computed tomography scanning Alexandra L. Matteia, Mark L. Ricciob, Frank W. Avilaa, and Mariana F. Wolfner Physiological changes in females during and after mating are triggered by seminal fluid components in conjunction with female-derived molecules. In insects, these changes include increased egg production, storage of sperm, and changes in muscle contraction within the reproductive tract (RT). Such postmating changes have been studied in dissected RT tissues, but understanding their coordination in vivo requires a holistic view of the tissues and their interrelationships. Here, we used high-resolution, multiscale micro-computed tomography (CT) scans to visualize and measure postmating changes in situ in the Drosophila female RT before, during, and after mating. These studies reveal previously unidentified dynamic changes in the conformation of the female RT that occur after mating. Our results also reveal how the reproductive organs temporally shift in concert within the confines of the abdomen. For example, we observed chiral loops in the uterus and in the upper common oviduct that relax and constrict throughout sperm storage and egg movement. We found that specific seminal fluid proteins or female secretions mediate some of the postmating changes in morphology. The morphological movements, in turn, can cause further changes due to the connections among organs. In addition, we observed apparent copulatory damage to the female intima, suggesting a mechanism for entry of seminal proteins, or other exogenous components, into the female’s circulatory system. The 3D reconstructions provided by high-resolution micro-CT scans reveal how male and female molecules and anatomy interface to carry out and coordinate mating-dependent changes in the female’s reproductive physiology. Science Science 5 June 2015: Vol. 348 no. 6239 pp. 1139-1143 DOI: 10.1126/science.aaa4788 Genomic signatures of evolutionary transitions from solitary to group living Karen M. Kapheim1,2,3,*,†, Hailin Pan4,*, Cai Li4,5, Steven L. Salzberg6,7, Daniela Puiu7, Tanja Magoc7, Hugh M. Robertson1,2, Matthew E. Hudson1,8, Aarti Venkat1,8,9, Brielle J. Fischman1,10,11, Alvaro Hernandez12, Mark Yandell13,14, Daniel Ence13, Carson Holt13,14, George D. Yocum15, William P. Kemp15, Jordi Bosch16, Robert M. Waterhouse17,18,19,20, Evgeny M. Zdobnov17,18, Eckart Stolle21,22, F. Bernhard Kraus21,23, Sophie Helbing21, Robin F. A. Moritz21,24, Karl M. Glastad25, Brendan G. Hunt26, Michael A. D. Goodisman25, Frank Hauser27, Cornelis J. P. Grimmelikhuijzen27, Daniel Guariz Pinheiro28,29, Francis Morais Franco Nunes30, Michelle Prioli Miranda Soares28, Érica Donato Tanaka31, Zilá Luz Paulino Simões28, Klaus Hartfelder32, Jay D. Evans33, Seth M. Barribeau34, Reed M. Johnson35, Jonathan H. Massey2,36, Bruce R. Southey37, Martin Hasselmann38, Daniel Hamacher38, Matthias Biewer38, Clement F. Kent39,40, Amro Zayed39, Charles Blatti III1,41, Saurabh Sinha1,41, J. Spencer Johnston42, Shawn J. Hanrahan42, Sarah D. Kocher43, Jun Wang4,44,45,46,47,†, Gene E. Robinson1,48,†, Guojie Zhang4,49,† The evolution of eusociality is one of the major transitions in evolution, but the underlying genomic changes are unknown. We compared the genomes of 10 bee species that vary in social complexity, representing multiple independent transitions in social evolution, and report three major findings. First, many important genes show evidence of neutral evolution as a consequence of relaxed selection with increasing social complexity. Second, there is no single road map to eusociality; independent evolutionary transitions in sociality have independent genetic underpinnings. Third, though clearly independent in detail, these transitions do have similar general features, including an increase in constrained protein evolution accompanied by increases in the potential for gene regulation and decreases in diversity and abundance of transposable elements. Eusociality may arise through different mechanisms each time, but would likely always involve an increase in the complexity of gene networks. Science 12 June 2015: Vol. 348 no. 6240 pp. 1268-1270 DOI: 10.1126/science.aaa2850 A male-determining factor in the mosquito Aedes aegypti Andrew Brantley Hall1,2,3,*, Sanjay Basu3,4,*, Xiaofang Jiang1,2,3, Yumin Qi2,3, Vladimir A. Timoshevskiy3,4, James K. Biedler2,3, Maria V. Sharakhova3,4, Rubayet Elahi2, Michelle A. E. Anderson3,4, Xiao-Guang Chen5, Igor V. Sharakhov1,3,4, Zach N. Adelman1,3,4,†, Zhijian Tu1,2,3,† Sex determination in the mosquito Aedes aegypti is governed by a dominant male-determining factor (M factor) located within a Y chromosome–like region called the M locus. Here, we show that an M-locus gene, Nix, functions as an M factor in A. aegypti. Nix exhibits persistent M linkage and early embryonic expression, two characteristics required of an M factor. Nix knockout with clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 resulted in largely feminized genetic males and the production of female isoforms of two key regulators of sexual differentiation: doublesex and fruitless. Ectopic expression of Nix resulted in genetic females with nearly complete male genitalia. Thus, Nix is both required and sufficient to initiate male development. This study provides a foundation for mosquito control strategies that convert female mosquitoes into harmless males. Science 26 June 2015: Vol. 348 no. 6242 pp. 1477-1481 DOI: 10.1126/science.aab1452 A Cas9–guide RNA complex preorganized for target DNA recognition Fuguo Jiang1, Kaihong Zhou2, Linlin Ma2, Saskia Gressel3, Jennifer A. Doudna1,2,4,5,6,7,* Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA “seed” sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition–competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a “seed” mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes. Symmetry Symmetry 2015, 7(2), 843-934; doi:10.3390/sym7020843 Analyzing Fluctuating Asymmetry with Geometric Morphometrics: Concepts, Methods, and Applications Christian Peter Klingenberg Approximately two decades after the first pioneering analyses, the study of shape asymmetry with the methods of geometric morphometrics has matured and is a burgeoning field. New technology for data collection and new methods and software for analysis are widely available and have led to numerous applications in plants and animals, including humans. This review summarizes the concepts and morphometric methods for studying asymmetry of shape and size. After a summary of mathematical and biological concepts of symmetry and asymmetry, a section follows that explains the methods of geometric morphometrics and how they can be used to analyze asymmetry of biological structures. Geometric morphometric analyses not only tell how much asymmetry there is, but also provide information about the patterns of covariation in the structure under study. Such patterns of covariation in fluctuating asymmetry can provide valuable insight about the developmental basis of morphological integration, and have become important tools for evolutionary developmental biology. The genetic basis of fluctuating asymmetry has been studied from empirical and theoretical viewpoints, but serious challenges remain in this area. There are many promising areas for further research that are only little explored at present.