A reference quality genome assembly for the Jewel scarab Chrysina gloriosa.

The jewel scarab Chrysina gloriosa is one of the most charismatic beetles in the United States and is found from the mountains of West Texas to the Southeastern Arizona sky islands. This species is highly sought by professional and amateur collectors worldwide due to its gleaming metallic coloration. However, the impact of the large-scale collection of this beetle on its populations is unknown, and there is a limited amount of genetic information available to make informed decisions about its conservation. As a first step, we present the genome of C. gloriosa, which we reconstructed using a single female specimen sampled from our ongoing effort to document population connectivity and the demographic history of this beetle. Using a combination of long-read sequencing and Omni-C data, we reconstructed the C. gloriosa genome at a near-chromosome level. Our genome assembly consisted of 454 scaffolds spanning 642 MB, with the ten largest scaffolds capturing 98% of the genome. The scaffold N50 was 72 MB, and the BUSCO score was 95.5%. This genome assembly will be an essential tool to accelerate understanding C. gloriosa biology and help make informed decisions for the conservation of Chrysina and other species with similar distributions in this region. This genome assembly will further serve as a community resource for comparative genomic analysis.

Diptera and Drosophila Karyotype Databases: A Useful Dataset to Guide Evolutionary and Genomic Studies.

Karyotypes and chromosome data have been widely used in many subfields of biology over the last century. Unfortunately, this data is largely scattered among hundreds of articles, books, and theses, many of which are only available behind paywalls. This creates a barrier to new researchers wishing to use this data, especially those from smaller institutions or in countries lacking institutional access to much of the scientific literature. We solved this problem by building two datasets for true flies (Order: Diptera and one specific to Drosophila), These datasets are available via a public interactive database that allows users to explore, visualize and download all data. The Diptera karyotype databases currently contain a total of 3,474 karyotype records from 538 publications. Synthesizing this data, we show several groups are of particular interest for future investigations by whole genome sequencing.

Identification and Characterization of Small RNA Markers of Age in the Blow Fly Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae).

Blow fly development is important in decomposition ecology, agriculture, and forensics. Much of the impact of these species is from immature samples, thus knowledge of their development is important to enhance or ameliorate their effects. One application of this information is the estimation of immature insect age to provide temporal information for death investigations. While traditional markers of age such as stage and size are generally accurate, they lack precision in later developmental stages. We used miRNA sequencing to measure miRNA expression, throughout development, of the secondary screwworm, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae) and identified 217 miRNAs present across the samples. Ten were identified to be significantly differentially expressed in larval samples and seventeen were found to be significantly differentially expressed in intrapuparial samples. Twenty-eight miRNAs were identified to be differentially expressed between sexes. Expression patterns of two miRNAs, miR-92b and bantam, were qPCR-validated in intrapuparial samples; these and likely food-derived miRNAs appear to be stable markers of age in C. macellaria. Our results support the use of miRNAs for developmental markers of age and suggest further investigations across species and under a range of abiotic and biotic conditions.

Special Issue: Genetic Basis of Phenotypic Variation in Drosophila and Other Insects.

Next-generation sequencing provides a nearly complete genomic sequence for model and non-model species alike; however, this wealth of sequence data includes no road map [...].

Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes.

The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

The probability of fusions joining sex chromosomes and autosomes.

Chromosome fusion and fission are primary mechanisms of karyotype evolution. In particular, the fusion of a sex chromosome and an autosome has been proposed as a mechanism to resolve intralocus sexual antagonism. If sexual antagonism is common throughout the genome, we should expect to see an excess of fusions that join sex chromosomes and autosomes. Here, we present a null model that provides the probability of a sex chromosome autosome fusion, assuming all chromosomes have an equal probability of being involved in a fusion. This closed-form expression is applicable to both male and female heterogametic sex chromosome systems and can accommodate unequal proportions of fusions originating in males and females. We find that over 25% of all chromosomal fusions are expected to join a sex chromosome and an autosome whenever the diploid autosome count is fewer than 16, regardless of the sex chromosome system. We also demonstrate the utility of our model by analysing two contrasting empirical datasets: one from Drosophila and one from the jumping spider genus Habronattus. We find that in the case of Habronattus, there is a significant excess of sex chromosome autosome fusions but that in Drosophila there are far fewer sex chromosome autosome fusions than would be expected under our null model.

Lineage-specific patterns of chromosome evolution are the rule not the exception in Polyneoptera insects.

The structure of a genome can be described at its simplest by the number of chromosomes and the sex chromosome system it contains. Despite over a century of study, the evolution of genome structure on this scale remains recalcitrant to broad generalizations that can be applied across clades. To address this issue, we have assembled a dataset of 823 karyotypes from the insect group Polyneoptera. This group contains orders with a range of variations in chromosome number, and offer the opportunity to explore the possible causes of these differences. We have analysed these data using both phylogenetic and taxonomic approaches. Our analysis allows us to assess the importance of rates of evolution, phylogenetic history, sex chromosome systems, parthenogenesis and genome size on variation in chromosome number within clades. We find that fusions play a key role in the origin of new sex chromosomes, and that orders exhibit striking differences in rates of fusions, fissions and polyploidy. Our results suggest that the difficulty in finding consistent rules that govern evolution at this scale may be due to the presence of many interacting forces that can lead to variation among groups.

An Evaluation of Differentially Spliced Genes as Markers of Sex for Forensic Entomology,.

Blow flies (Calliphoridae) are important medically and economically and are commonly used in forensics as temporal markers in death investigations. While phenotypic traits in adult flies can be sexually dimorphic, sex identification in immatures is difficult. Consequently, little is known about how sex may result in developmental disparities among sexes even though there are indications that they may be important in some instances. Since genetic mechanisms for sex are well studied in model flies and species of agricultural and medical importance, we exploit the sex-specifically spliced genes transformer (tra) and doublesex (dsx) in the sex determination pathway to optimize a sex identification assay for immatures. Using known primer sets for tra and with a novel one for dsx, we develop PCR assays for identifying sex in four forensically relevant Calliphoridae species: Lucilia sericata (Meigen), Lucilia cuprina (Wiedemann), Cochliomyia macellaria (Fabricius), and Chrysomya rufifacies (Macquart) and evaluated their performance. Band detection rates were found to range from 71 to 100%, call rates ranged from 90 to 100%, and no error was found when bands could be called. Such information is informative for purposes of testimony and in preparation for development studies. The developed assays will assist in further differentiating sexually dimorphic differences in development of the Calliphoridae and aid in more accurately estimating insect age when age predictive markers (size, development time, molecular expression) are sexually dimorphic.

Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA.

Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. We measure underreplication in 132 species across the Drosophila genus and leverage these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented here estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila.

Flying High-Muscle-Specific Underreplication in Drosophila.

Drosophila underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the Drosophila salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. Our question was, why half? To address this question, we use flow cytometry to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, we find that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control.

Effect of Phenotype Selection on Genome Size Variation in Two Species of Diptera.

Genome size varies widely across organisms yet has not been found to be related to organismal complexity in eukaryotes. While there is no evidence for a relationship with complexity, there is evidence to suggest that other phenotypic characteristics, such as nucleus size and cell-cycle time, are associated with genome size, body size, and development rate. However, what is unknown is how the selection for divergent phenotypic traits may indirectly affect genome size. Drosophila melanogaster were selected for small and large body size for up to 220 generations, while Cochliomyia macellaria were selected for 32 generations for fast and slow development. Size in D. melanogaster significantly changed in terms of both cell-count and genome size in isolines, but only the cell-count changed in lines which were maintained at larger effective population sizes. Larger genome sizes only occurred in a subset of D. melanogaster isolines originated from flies selected for their large body size. Selection for development time did not change average genome size yet decreased the within-population variation in genome size with increasing generations of selection. This decrease in variation and convergence on a similar mean genome size was not in correspondence with phenotypic variation and suggests stabilizing selection on genome size in laboratory conditions.

Genome Size Evolution Differs Between Drosophila Subgenera with Striking Differences in Male and Female Genome Size in Sophophora.

Genome size varies across the tree of life, with no clear correlation to organismal complexity or coding sequence, but with differences in non-coding regions. Phylogenetic methods have recently been incorporated to further disentangle this enigma, yet most of these studies have focused on widely diverged species. Few have compared patterns of genome size change in closely related species with known structural differences in the genome. As a consequence, the relationship between genome size and differences in chromosome number or inter-sexual differences attributed to XY systems are largely unstudied. We hypothesize that structural differences associated with chromosome number and X-Y chromosome differentiation, should result in differing rates and patterns of genome size change. In this study, we utilize the subgenera within the Drosophila to ask if patterns and rates of genome size change differ between closely related species with differences in chromosome numbers and states of the XY system. Genome sizes for males and females of 152 species are used to answer these questions (with 92 newly added or updated estimates). While we find no relationship between chromosome number and genome size or chromosome number and inter-sexual differences in genome size, we find evidence for differing patterns of genome size change between the subgenera, and increasing rates of change throughout time. Estimated shifts in rates of change in sex differences in genome size occur more often in Sophophora and correspond to known neo-sex events.

Genome Size Evolution within and between the Sexes.

Genome sizes are known to vary between closely related species, but the patterns behind this variation have yet to be fully understood. Although this variation has been evaluated between species and within sexes, unknown is the extent to which this variation is driven by differentiation in sex chromosomes. To address this longstanding question, we examine the mode and tempo of genome size evolution for a total of 87 species of Drosophilidae, estimating and updating male genome size values for 44 of these species. We compare the evolution of genome size within each sex to the evolution of the differences between the sexes. Utilizing comparative phylogenetic methods, we find that male and female genome size evolution is largely a neutral process, reflective of phylogenetic relatedness between species, which supports the newly proposed accordion model for genome size change. When similarly analyzed, the difference between the sexes due to heteromorphic sex chromosomes is a dynamic process; the male-female genome size difference increases with time with or without known neo-Y events or complete loss of the Y. Observed instances of rapid change match theoretical expectations and known neo-Y and Y loss events in individual species.

Genome Size Estimation and Quantitative Cytogenetics in Insects.

With care, it is possible using flow cytometry to create a precise and accurate estimate of the genome size of an insect that is useful for genomics, genetics, molecular/cell biology, or systematics. Genome size estimation is a useful first step in a complete genome sequencing project. The number of sequencing reads required to produce a given level of coverage depends directly upon the 1C amount of DNA per cell, while an even more critical need is an accurate 1C genome size estimate to compare against the final assembly. Here we present a detailed protocol to estimate genome size using flow cytometry. Published genome size estimates should be submitted to genomesize.com so that they are available to all.

Genome Size in North American Fireflies: Substantial Variation Likely Driven by Neutral Processes.

Eukaryotic genomes show tremendous size variation across taxa. Proximate explanations for genome size variation include differences in ploidy and amounts of noncoding DNA, especially repetitive DNA. Ultimate explanations include selection on physiological correlates of genome size such as cell size, which in turn influence body size, resulting in the often-observed correlation between body size and genome size. In this study, we examined body size and repetitive DNA elements in relationship to the evolution of genome size in North American representatives of a single beetle family, the Lampyridae (fireflies). The 23 species considered represent an excellent study system because of the greater than 5-fold range of genome sizes, documented here using flow cytometry, and the 3-fold range in body size, measured using pronotum width. We also identified common genomic repetitive elements using low-coverage sequencing. We found a positive relationship between genome size and repetitive DNA, particularly retrotransposons. Both genome size and these elements were evolving as expected given phylogenetic relatedness. We also tested whether genome size varied with body size and found no relationship. Together, our results suggest that genome size is evolving neutrally in fireflies.

The mode and tempo of genome size evolution in the subgenus Sophophora.

Genome size varies widely across organisms, with no apparent tie to organismal complexity. While genome size is inherited, there is no established evolutionary model for this trait. Hypotheses have been postulated for the observed variation in genome sizes across species, most notably the effective population size hypothesis, the mutational equilibrium hypothesis, and the adaptive hypothesis. While much data has been collected on genome size, the above hypotheses have largely ignored impacts from phylogenetic relationships. In order to test these competing hypotheses, genome sizes of 87 Sophophora species were analyzed in a comparative phylogenetic approach using Pagel's parameters of evolution, Blomberg's K, Abouheif's Cmean and Moran's I. In addition to testing the mode and rate of genome size evolution in Sophophora species, the effect of number of taxa on detection of phylogenetic signal was analyzed for each of these comparative phylogenetic methods. Sophophora genome size was found to be dependent on the phylogeny, indicating that evolutionary time was important for predicting the variation among species. Genome size was found to evolve gradually on branches of the tree, with a rapid burst of change early in the phylogeny. These results suggest that Sophophora genome size has experienced gradual changes, which support the largely theoretical mutational equilibrium hypothesis. While some methods (Abouheif's Cmean and Moran's I) were found to be affected by increasing taxa numbers, more commonly used methods (λ and Blomberg's K) were found to have increasing reliability with increasing taxa number, with significantly more support with fifteen or more taxa. Our results suggest that these comparative phylogenetic methods, with adequate taxon sampling, can be a powerful way to uncover the enigma that is genome size variation through incorporation of phylogenetic relationships.

Genome size correlates with reproductive fitness in seed beetles.

The ultimate cause of genome size (GS) evolution in eukaryotes remains a major and unresolved puzzle in evolutionary biology. Large-scale comparative studies have failed to find consistent correlations between GS and organismal properties, resulting in the 'C-value paradox'. Current hypotheses for the evolution of GS are based either on the balance between mutational events and drift or on natural selection acting upon standing genetic variation in GS. It is, however, currently very difficult to evaluate the role of selection because within-species studies that relate variation in life-history traits to variation in GS are very rare. Here, we report phylogenetic comparative analyses of GS evolution in seed beetles at two distinct taxonomic scales, which combines replicated estimation of GS with experimental assays of life-history traits and reproductive fitness. GS showed rapid and bidirectional evolution across species, but did not show correlated evolution with any of several indices of the relative importance of genetic drift. Within a single species, GS varied by 4-5% across populations and showed positive correlated evolution with independent estimates of male and female reproductive fitness. Collectively, the phylogenetic pattern of GS diversification across and within species in conjunction with the pattern of correlated evolution between GS and fitness provide novel support for the tenet that natural selection plays a key role in shaping GS evolution.

Endopolyploidy changes with age-related polyethism in the honey bee, Apis mellifera.

Honey bees (Apis mellifera) exhibit age polyethism, whereby female workers assume increasingly complex colony tasks as they age. While changes in DNA methylation accompany age polyethism, other DNA modifications accompanying age polyethism are less known. Changes in endopolyploidy (DNA amplification in the absence of cell division) with increased larval age are typical in many insect cells and are essential in adults for creating larger cells, more copies of essential loci, or greater storage capacity in secretory cells. However, changes in endopolyploidy with increased adult worker age and polyethism are unstudied. In this study, we examined endopolyploidy in honey bee workers ranging in age from newly emerged up to 55 days old. We found a nonsignificant increase in ploidy levels with age (P < 0.1) in the most highly endopolyploid secretory cells, the Malpighian tubules. All other cell types decreased ploidy levels with age. Endopolyploidy decreased the least amount (nonsignificant) in neural (brain) cells and the stinger (P < 0.1). There was a significant reduction of endopolyploidy with age in leg (P < 0.05) and thoracic (P < 0.001) muscles. Ploidy in thoracic muscle dropped from an average of 0.5 rounds of replication in newly emerged workers to essentially no rounds of replication (0.125) in the oldest workers. Ploidy reduction in flight muscle cells is likely due to the production of G1 (2C) nuclei by amitotic division in the multinucleate striated flight muscles that are essential to foragers, the oldest workers. We suggest that ploidy is constrained by the shape, size and makeup of the multinucleate striated muscle cells. Furthermore, the presence of multiple 2C nuclei might be optimal for cell function, while higher ploidy levels might be a dead-end strategy of some aging adult tissues, likely used to increase cell size and storage capacity in secretory cells.

Intrapopulation genome size variation in D. melanogaster reflects life history variation and plasticity.

We determined female genome sizes using flow cytometry for 211 Drosophila melanogaster sequenced inbred strains from the Drosophila Genetic Reference Panel, and found significant conspecific and intrapopulation variation in genome size. We also compared several life history traits for 25 lines with large and 25 lines with small genomes in three thermal environments, and found that genome size as well as genome size by temperature interactions significantly correlated with survival to pupation and adulthood, time to pupation, female pupal mass, and female eclosion rates. Genome size accounted for up to 23% of the variation in developmental phenotypes, but the contribution of genome size to variation in life history traits was plastic and varied according to the thermal environment. Expression data implicate differences in metabolism that correspond to genome size variation. These results indicate that significant genome size variation exists within D. melanogaster and this variation may impact the evolutionary ecology of the species. Genome size variation accounts for a significant portion of life history variation in an environmentally dependent manner, suggesting that potential fitness effects associated with genome size variation also depend on environmental conditions.

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.