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A massive adaptive radiation on the Hawaiian archipelago has produced approximately one quarter of the fly species in the family Drosophilidae. The Hawaiian Drosophila clade has long been recognized as a model system for the study of both the ecology of island endemics and the evolution of developmental mechanisms, but relatively few genomic and transcriptomic datasets are available for this group. We present here a differential expression analysis of the transcriptional profiles of two highly conserved embryonic stages in the Hawaiian picture-wing fly Drosophila grimshawi. When we compared our results to previously published datasets across the family Drosophilidae, we identified cases of both gains and losses of gene representation in D. grimshawi, including an apparent delay in Hox gene activation. We also found high expression of unannotated genes. Most transcripts of unannotated genes with open reading frames do not have homologs in non-Hawaiian Drosophila species, although the vast majority have sequence matches in other genomes of the Hawaiian picture-wing flies. Some of these genes may have arisen from non-coding sequence in the ancestor of Hawaiian flies or during the evolution of the clade. Our results suggests that both the modified use of ancestral genes and the evolution of new ones may occur in rapid radiations.RESEARCH HIGHLIGHTSThe early embryonic transcriptome of the Hawaiian fly Drosophila grimshawi shows a loss of expression of conserved Stage 5 genes, including the Hox genesThe de novo evolution of embryonically expressed genes may be occurring in the Hawaiian Drosophila lineageAUTHORS’ STATEMENTThis paper is not being considered for publication elsewhere. This study formed part of Madeline Chenevert’s M.S. thesis.

Epithelia are highly dynamic tissues displaying various types of tissue rearrangements (Weliky and Oster, 1990; Taylor and Adler, 2008; Harris and Tepass, 2010; Lee

The model organism Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.

Motivation Transposable Elements (TEs) or jumping genes are DNA sequences that have an intrinsic capability to move within a host genome from one genomic location to another. Studies show that the presence of a TE within or adjacent to a functional gene may alter its expression. TEs can also cause an increase in the rate of mutation and can even mediate duplications and large insertions and deletions in the genome, promoting gross genetic rearrangements. The proper classification of identified jumping genes is important for analyzing their genetic and evolutionary effects. An effective classifier, which can explain the role of TEs in germline and somatic evolution more accurately, is needed. In this study, we examine the performance of a variety of machine learning (ML) techniques and propose a robust method, ClassifyTE, for the hierarchical classification of TEs with high accuracy, using a stacking-based ML method. Results We propose a stacking-based approach for the hierarchical classification of TEs. When trained on three different benchmark datasets, our proposed system achieved 4%, 10.68% and 10.13% average percentage improvement (using the hF measure) compared to several state-of-the-art methods. We developed an end-to-end automated hierarchical classification tool based on the proposed approach, ClassifyTE, to classify TEs up to the super-family level. We further evaluated our method on a new TE library generated by a homology-based classification method and found relatively high concordance at higher taxonomic levels. Thus, ClassifyTE paves the way for a more accurate analysis of the role of TEs. Availability and implementation The source code and data are available at https://github.com/manisa/ClassifyTE. Supplementary information Supplementary data are available at Bioinformatics online.

Motivation Recent research has uncovered roles for transposable elements (TEs) in multiple evolutionary processes, ranging from somatic evolution in cancer to putatively adaptive germline evolution across species. Most models of TE population dynamics, however, have not incorporated actual genome sequence data. The effect of site integration preferences of specific TEs on evolutionary outcomes and the effects of different selection regimes on TE dynamics in a specific genome are unknown. We present a stochastic model of LINE-1 (L1) transposition in human cancer. This system was chosen because the transposition of L1 elements is well understood, the population dynamics of cancer tumors has been modeled extensively, and the role of L1 elements in cancer progression has garnered interest in recent years. Results Our model predicts that L1 retrotransposition (RT) can play either advantageous or deleterious roles in tumor progression, depending on the initial lesion size, L1 insertion rate and tumor driver genes. Small changes in the RT rate or set of driver tumor-suppressor genes (TSGs) were observed to alter the dynamics of tumorigenesis. We found high variation in the density of L1 target sites across human protein-coding genes. We also present an analysis, across three cancer types, of the frequency of homozygous TSG disruption in wild-type hosts compared to those with an inherited driver allele. Availability and implementation Source code is available at https://github.com/atallah-lab/neoplastic-evolution. Contact jlebien@uno.edu Supplementary information Supplementary data are available at Bioinformatics online.

The earliest stages of animal development are controlled by maternally deposited mRNA transcripts and proteins. Once the zygote is able to transcribe its own genome, maternal transcripts are degraded, in a tightly regulated process known as the maternal to zygotic transition (MZT). While this process has been well-studied within model species, we have little knowledge of how the pools of maternal and zygotic transcripts evolve. To characterize the evolutionary dynamics and functional constraints on early embryonic expression, we created a transcriptomic dataset for 14 Drosophila species spanning over 50 million years of evolution, at developmental stages before and after the MZT, and compared our results with a previously published Aedes aegypti developmental time course. We found deep conservation over 250 million years of a core set of genes transcribed only by the zygote. This select group is highly enriched in transcription factors that play critical roles in early development. However, we also identify a surprisingly high level of change in the transcripts represented at both stages over the phylogeny. While mRNA levels of genes with maternally deposited transcripts are more highly conserved than zygotic genes, those maternal transcripts that are completely degraded at the MZT vary dramatically between species. We also show that hundreds of genes have different isoform usage between the maternal and zygotic genomes. Our work suggests that maternal transcript deposition and early zygotic transcription are remarkably dynamic over evolutionary time, despite the widespread conservation of early developmental processes., Author summary Genetic control of embryonic development in all animals requires precise coordination between mother and zygote. The mother provides gene products to the egg to drive the earliest stages of development, until the zygote is able to transcribe its own genome. Many processes of early development are highly conserved over evolutionary time, and are critical for organism survival. Here, we determine how conserved the pools of transcripts provided by the mother and transcribed by the zygote are over evolutionary time, by sequencing all the transcripts at each stage in 14 species of fruit flies at different stages of development. We find a substantial conservation of transcripts represented, with the transcripts deposited by the mother being especially highly conserved. However, we also find considerable variation in these early transcript pools between species, suggesting that while early developmental processes are highly conserved, the gene products driving them may not be. This may be necessary for carrying out the processes of development in different environments.

The origin of new morphological structures requires the establishment of new genetic regulatory circuits to control their development, from initial specification to terminal differentiation. The upstream regulatory genes are usually the first to be identified, while the mechanisms that translate novel regulatory information into phenotypic diversity often remain obscure. In particular, elaborate sex-specific structures that have evolved in many animal lineages are inevitably controlled by sex-determining genes, but the genetic basis of sexually dimorphic cell differentiation is rarely understood. In this report, we examine the role of dachshund (dac), a gene with a deeply conserved function in sensory organ and appendage development, in the sex comb, a recently evolved male-specific structure found in some Drosophila species. We show that dac acts during metamorphosis to restrict sex comb development to the appropriate leg region. Localized repression of dac by the sex determination pathway is necessary for male-specific morphogenesis of sex comb bristles. This pupal function of dac is separate from its earlier role in leg patterning, and Dac at this stage is not dependent on the pupal expression of Distalless (Dll), the main regulator of dac during the larval period. Dll acts in the epithelial cells surrounding the sex comb during pupal development to promote sex comb rotation, a complex cellular process driven by coordinated cell rearrangement. Our results show that genes with well-conserved developmental functions can be re-used at later stages in development to regulate more recently evolved traits. This mode of gene co-option may be an important driver of evolutionary innovations.

On macroevolutionary time scales, the same genes can regulate the development of homologous structures through strikingly different cellular processes. Comparing the development of similar morphological traits in closely related species may help elucidate the evolutionary dissociation between pattern formation and morphogenesis. We address this question by focusing on the interspecific differences in sex comb development in Drosophilids. The sex comb is a recently evolved, male-specific structure composed of modified bristles. Previous work in the obscura and melanogaster species groups (Old World Sophophora) has identified two distinct cellular mechanisms that give rise to nearly identical adult morphologies. Here, we describe sex comb development in a species from a more distantly related lineage, the genus Lordiphosa. Although the expression of key regulatory genes is largely conserved in both clades, the cell behaviors responsible for sex comb formation show major differences between Old World Sophophora and Lordiphosa. We suggest that the many-to-one mapping between development and adult phenotype increases the potential for evolutionary innovations.

The Drosophila sex comb (SC) has been hailed as a powerful tool for integrative studies in development, evolution, and behavior, but its ontogeny is poorly understood, even in the model organism Drosophila melanogaster. Using 4D live imaging and other techniques, we carried out a detailed analysis of the cellular events that take place during the development of the SC. We showed that the comb and other contiguous bristle formations assemble from noncontiguous precursor cells, which join together through intercalation. Most of the rotation of the SC (which has a longitudinal orientation in D. melanogaster but is initially transverse) occurs after this stage, when the structure is a single unit. We have provided evidence that male-specific convergent extension through cell rearrangement is responsible for both this rotation and another sexually dimorphic bristle trait. Contiguous bristle formations act as barriers to cell movement within the epithelium, and we demonstrated that a particularly rapid rotation of the proximal region of the comb is associated with the presence of a constricted area between a portion of the SC and a transverse row of contiguous bristle precursors. Our results suggest that the cell dynamics in the neighborhood of the SC may have biased its evolution.

Summary The morphogenetic field, a fundamental concept of classical embryology, is once again being invoked to describe developmental processes. Because the evolution of adult structures requires the modification of development, the ways in which morphogenetic fields can change over time may yield insights into evolutionary possibilities. We considered how the duplication/multiplication of a morphogenetic field in fruit flies, caused by the previously described obake (obk) mutation, is regulated by genetic and environmental factors. Mutations of genes in the canonical antenna-producing imaginal disc pathway suppressed duplication as expected, although the results suggested that other pathways might also be involved. Overgrowth mutations, expected to increase duplication, actually suppressed it. Mutations in the heat-shock protein gene Hsp83 did not uniformly enhance obk expressivity as hypothesized. Using third chromosomes extracted from wild-derived lines, natural genetic variation for modifiers of obk function was found to be extensive. Larval crowding suppressed the obk phenotype, but there was no evidence of trade-offs between body or head size and arista number. Our results suggest that a complex interplay of genetic and environmental factors in the regulation of fields may be responsible for ample natural variation in the expressivity of adult phenotypes, affording multiple opportunities for selection and evolutionary modification.

In spite of the diversity of possible biological forms observed in nature, a limited range of morphospace is frequently occupied for a given trait. Several mechanisms have been proposed to explain this bias in the distribution of phenotypes including selection, drift, and developmental constraints. Despite extensive work on phenotypic bias, the underlying developmental mechanisms explaining why particular regions of morphological space remain unoccupied are poorly understood. To address this issue, we studied the sex comb, a group of modified bristles used in courtship that shows marked morphological diversity among Drosophila species. In many Drosophila species including Drosophila melanogaster, the sex comb rotates 90° to a vertical position during development. Here we analyze the effect of changing D. melanogaster sex comb length on the process of rotation. We find that artificial selection changes the number of bristles per comb without a proportional change in the space available for rotation. As a result, when increasing sex comb length, rather than displaying a similar straight vertical shape observed in other Drosophila species, long sex combs bend because rotation is blocked by a neighboring row of bristles. Our results show ways in which morphologies that would be favored by natural selection are apparently impossible to achieve developmentally. These findings highlight the potential role of development in modifying selectable variation in the evolution of Drosophila sex comb length.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community., National Institutes of Health (U.S.) (NIDDK (DK015600-18)), National Institutes of Health (U.S.) (Extramural program (1ROIGM082843)), National Institutes of Health (U.S.) (Extramural program (U01HB004271))

Evolutionary innovation can allow a species access to a new ecological niche, potentially reducing competition with closely related species. While the vast majority ofDrosophilaflies feed on rotting fruit and other decaying matter, and are harmless to human activity,Drosophila suzukii, which has a morphologically modified ovipositor, is capable of colonizing live fruit that is still in the process of ripening, causing massive agricultural damage. Here, we conducted the first comparative analysis of this species and its close relatives, analysing both ovipositor structure and fruit susceptibility. We found that the ovipositor of the species most closely related toD. suzukii,Drosophila subpulchrella, has a similar number of enlarged, evolutionarily derived bristles, but a notably different overall shape. LikeD. suzukii,D. subpulchrellaflies are capable of puncturing the skin of raspberries and cherries, but we found no evidence that they could penetrate the thicker skin of two varieties of grapes. More distantly related species, one of which has previously been mistaken forD. suzukii, have blunt ovipositors with small bristles. While they did not penetrate fruit skin in any of the assays, they readily colonized fruit interiors where the skin was broken. Our results suggest that considering evolutionary context may be beneficial to the management of invasive species.

The sequencing depth necessary for documenting differential gene expression using RNA-Seq has been little explored outside of model systems. In particular, the depth required to analyze large-scale patterns of differential transcription factor expression is not known. The goal of the present study is to explore the effectiveness of shallow (relatively low read depth) RNA-Seq. We focus on two tissues in the honey bee: the sting gland and the digestive tract. The sting gland is an experimentally well-understood tissue that we use to benchmark the utility of this approach. We use the digestive tract to test the results obtained with the sting gland, and to conduct RNA-Seq between tissue types. Using a list of experimentally verified genes conferring tissue-specific functions in the sting gland, we show that relatively little read depth is necessary to identify them. We argue that this result should be broadly applicable, since genes important for tissue-specific functions often have robust expression patterns, and because we obtained similar results in our analysis of the digestive tract. Furthermore, we demonstrate that the differential expression of transcription factors, which are transcribed at low levels compared to other genes, can nevertheless often be determined using shallow RNA-Seq. Overall, we find over 150 differentially expressed transcription factors in our tissues at a read depth of only 12 million. This work shows the utility of low-depth sequencing for identifying genes important for tissue-specific functions. It also verifies the often-held belief that transcription factors show low levels of expression, while demonstrating that, in spite of this, they are frequently amenable to shallow RNA-Seq. Our findings should be of benefit to researchers using RNA-Seq in many different biological systems.

Background A composite biological structure, such as an insect head or abdomen, contains many internal structures with distinct functions. Composite structures are often used in RNA-seq studies, though it is unclear how expression of the same gene in different tissues and structures within the same structure affects the measurement (or even utility) of the resulting patterns of gene expression. Here we determine how complex composite tissue structure affects measures of gene expression using RNA-seq. Results We focus on two structures in the honey bee (the sting gland and digestive tract) both contained within one larger structure, the whole abdomen. For each of the three structures, we used RNA-seq to identify differentially expressed genes between two developmental stages, nurse bees and foragers. Based on RNA-seq for each structure-specific extraction, we found that RNA-seq with composite structures leads to many false negatives (genes strongly differentially expressed in particular structures which are not found to be differentially expressed within the composite structure). We also found a significant number of genes with one pattern of differential expression in the tissue-specific extraction, and the opposite in the composite extraction, suggesting multiple signals from such genes within the composite structure. We found these patterns for different classes of genes including transcription factors. Conclusions Many RNA-seq studies currently use composite extractions, and even whole insect extractions, when tissue and structure specific extractions are possible. This is due to the logistical difficultly of micro-dissection and unawareness of the potential errors associated with composite extractions. The present study suggests that RNA-seq studies of composite structures are prone to false negatives and difficult to interpret positive signals for genes with variable patterns of local expression. In general, our results suggest that RNA-seq on large composite structures should be avoided unless it is possible to demonstrate that the effects shown here do not exist for the genes of interest.

Whether coding or regulatory sequence change is more important to the evolution of phenotypic novelty is one of biology’s major unresolved questions. The field of evo–devo has shown that in early development changes to regulatory regions are the dominant mode of genetic change, but whether this extends to the evolution of novel phenotypes in the adult organism is unclear. Here, we conduct ten RNA-Seq experiments across both novel and conserved tissues in the honey bee to determine to what extent postdevelopmental novelty is based on changes to the coding regions of genes. We make several discoveries. First, we show that with respect to novel physiological functions in the adult animal, positively selected tissue-specific genes of high expression underlie novelty by conferring specialized cellular functions. Such genes are often, but not always taxonomically restricted genes (TRGs). We further show that positively selected genes, whether TRGs or conserved genes, are the least connected genes within gene expression networks. Overall, this work suggests that the evo–devo paradigm is limited, and that the evolution of novelty, postdevelopment, follows additional rules. Specifically, evo–devo stresses that high network connectedness (repeated use of the same gene in many contexts) constrains coding sequence change as it would lead to negative pleiotropic effects. Here, we show that in the adult animal, the converse is true: Genes with low network connectedness (TRGs and tissue-specific conserved genes) underlie novel phenotypes by rapidly changing coding sequence to perform new-specialized functions.

On macroevolutionary time scales, the same genes can regulate the development of homologous structures through strikingly different cellular processes. Comparing the development of similar morphological traits in closely related species may help elucidate the evolutionary dissociation between pattern formation and morphogenesis. We address this question by focusing on the interspecific differences in sex comb development in Drosophilids. The sex comb is a recently evolved, male-specific structure composed of modified bristles. Previous work in the obscura and melanogaster species groups (Old World Sophophora) has identified two distinct cellular mechanisms that give rise to nearly identical adult morphologies. Here, we describe sex comb development in a species from a more distantly related lineage, the genus Lordiphosa. Although the expression of key regulatory genes is largely conserved in both clades, the cell behaviors responsible for sex comb formation show major differences between Old World Sophophora and Lordiphosa. We suggest that the many-to-one mapping between development and adult phenotype increases the potential for evolutionary innovations.

The quest to understand the relationship between an organism's DNA sequence and three-dimensional form is an interdisciplinary task, integrating diverse fields of the life sciences. The relevance of the metaphor of a genotype-phenotype map is explored from a developmental perspective, in light of the recent concept of a "molecular toolkit" of protein-coding genes, and the widespread view that analyzing the logic and mechanics of gene regulation at multiple levels is key to explaining how morphology is genetically encoded. We discuss the challenges of decoding genomes despite variable genetic backgrounds, the dynamically changing physical and molecular contexts of the internal environment during development, and the impact of external forces on morphogenesis.

The most complex and diverse secondary sexual character in Drosophila is the sex comb (SC), an arrangement of modified bristles on the forelegs of a subclade of male fruit flies. We examined SC formation in six representative nonmodel fruit fly species, in an effort to understand how the variation in comb patterning arises. We first compared SC development in two species with relatively small combs, Drosophila takahashii, where the SCs remain approximately transverse, and Drosophila biarmipes, where two rows of SC teeth rotate and move in an anterior direction relative to other bristle landmarks. We then analyzed comb ontogeny in species with prominent extended SCs parallel to the proximodistal axis, including Drosophila ficusphila and species of the montium subgroup. Our study allowed us to identify two general methods of generating longitudinal combs on the tarsus, and we showed that a montium subgroup species (Drosophila nikananu) with a comb convergently similar in size, orientation and position to the model organism Drosophila melanogaster, forms its SC through a different developmental mechanism. We also found that the protein product of the leg patterning gene, dachshund (dac), is strongly reduced in the SC in all species, but not in other bristles. Our results suggest that an apparent constraint on SC position in the adult may be attributable to at least two different lineage-specific developmental processes, although external forces could also play a role.

The quest to understand the relationship between an organism's DNA sequence and three-dimensional form is an interdisciplinary task, integrating diverse fields of the life sciences. The relevance of the metaphor of a genotype-phenotype map is explored from a developmental perspective, in light of the recent concept of a "molecular toolkit" of protein-coding genes, and the widespread view that analyzing the logic and mechanics of gene regulation at multiple levels is key to explaining how morphology is genetically encoded. We discuss the challenges of decoding genomes despite variable genetic backgrounds, the dynamically changing physical and molecular contexts of the internal environment during development, and the impact of external forces on morphogenesis.

SummaryEusocial behavior has arisen in few animal groups, most notably in the aculeate Hymenoptera, a clade comprising ants, bees, and stinging wasps [1–4]. Phylogeny is crucial to understanding the evolution of the salient features of these insects, including eusociality [5]. Yet the phylogenetic relationships among the major lineages of aculeate Hymenoptera remain contentious [6–12]. We address this problem here by generating and analyzing genomic data for a representative series of taxa. We obtain a single well-resolved and strongly supported tree, robust to multiple methods of phylogenetic inference. Apoidea (spheciform wasps and bees) and ants are sister groups, a novel finding that contradicts earlier views that ants are closer to ectoparasitoid wasps. Vespid wasps (paper wasps, yellow jackets, and relatives) are sister to all other aculeates except chrysidoids. Thus, all eusocial species of Hymenoptera are contained within two major groups, characterized by transport of larval provisions and nest construction, likely prerequisites for the evolution of eusociality. These two lineages are interpolated among three other clades of wasps whose species are predominantly ectoparasitoids on concealed hosts, the inferred ancestral condition for aculeates [2]. This phylogeny provides a new framework for exploring the evolution of nesting, feeding, and social behavior within the stinging Hymenoptera.