'Junk' that matters: the role of transposable elements in bumblebee genome evolution.

Transposable elements (TEs) are mobile DNA sequences that are widely distributed in eukaryotic genomes, where they are known to serve as a major force in genome evolution. The phenotypic impacts of TEs, while less well-studied, have also been discovered. Bumblebees are globally important pollinators in natural ecosystems and agriculture. Although TEs comprise a small fraction of bumblebee genomes, emerging evidence suggests that TEs are the major contributor of genome size variation across species and are involved in the formation of new coding and regulatory sequences. We review recent discoveries related to TEs in bumblebees, as well as outlining three key questions for the future of the field. In the future, we argue long-read sequencing technologies and genome editing techniques will help us identify TEs in bumblebees, unveil mechanisms that could account for their silencing and limited abundance, and uncover their contributions to phenotypic diversification, ecological adaptation, and speciation.

Thermal stress and mutation accumulation increase heat shock protein expression in Daphnia.

Understanding the short- and long-term consequences of climate change is a major challenge in biology. For aquatic organisms, temperature changes and drought can lead to thermal stress and habitat loss, both of which can ultimately lead to higher mutation rates. Here, we examine the effect of high temperature and mutation accumulation on gene expression at two loci from the heat shock protein (HSP) gene family, HSP60 and HSP90. HSPs have been posited to serve as 'mutational capacitors' given their role as molecular chaperones involved in protein folding and degradation, thus buffering against a wide range of cellular stress and destabilization. We assayed changes in HSP expression across 5 genotypes of Daphnia magna, a sentinel species in ecology and environmental biology, with and without acute exposure to thermal stress and accumulated mutations. Across genotypes, HSP expression increased ~ 6× in response to heat and ~ 4× with mutation accumulation, individually. Both factors simultaneously (lineages with high mutation loads exposed to high heat) increased gene expression ~ 23×-much more than that predicted by an additive model. Our results corroborate suggestions that HSPs can buffer against not only the effects of heat, but also mutations-a combination of factors both likely to increase in a warming world. Supplementary Information: The online version contains supplementary material available at 10.1007/s10682-022-10209-1.

Estimating somatic mutation rates by bottlenecked duplex sequencing in non-model organisms: Daphnia magna as a case study.

Somatic mutations are evolutionarily important as determinants of individual organismal fitness, as well as being a focus of clinical research on age-related disease, such as cancer. Identifying somatic mutations and quantifying mutation rates, however, is extremely challenging and genome-wide somatic mutation rates have only been reported for a few model organisms. Here, we describe the application of Duplex Sequencing on bottlenecked WGS libraries to quantify somatic nuclear genome-wide base substitution rates in Daphnia magna. Daphnia, historically an ecological model system, has more recently been the focus of mutation studies, in part because of its high germline mutation rates. Using our protocol and pipeline, we estimate a somatic mutation rate of 5.6 × 10-7 substitutions per site (in a genotype where the germline rate is 3.60 × 10-9 substitutions per site per generation). To obtain this estimate, we tested multiple dilution levels to maximize sequencing efficiency and developed bioinformatic filters needed to minimize false positives when a high-quality reference genome is not available. In addition to laying the groundwork for estimating genotypic variation in rates of somatic mutations within D. magna, we provide a framework for quantifying somatic mutations in other non-model systems, and also highlight recent innovations to single molecule sequencing that will help to further refine such estimates.

Intraspecific Variation in the Rates of Mutations Causing Structural Variation in Daphnia magna.

Mutations that cause structural variation are important sources of genetic variation upon which other evolutionary forces can act, however, they are difficult to observe and therefore few direct estimates of their rate and spectrum are available. Understanding mutation rate evolution, however, requires adding to the limited number of species for which direct estimates are available, quantifying levels of intraspecific variation in mutation rates, and assessing whether rate estimates co-vary across types of mutation. Here, we report structural variation-causing mutation rates (svcMRs) for six categories of mutations (short insertions and deletions, long deletions and duplications, and deletions and duplications at copy number variable sites) from nine genotypes of Daphnia magna collected from three populations in Finland, Germany, and Israel using a mutation accumulation approach. Based on whole-genome sequence data and validated using simulations, we find svcMRs are high (two orders of magnitude higher than base substitution mutation rates measured in the same lineages), highly variable among populations, and uncorrelated across categories of mutation. Furthermore, to assess the impact of scvMRs on the genome, we calculated rates while adjusting for the lengths of events and ran simulations to determine if the mutations occur in genic regions more or less frequently than expected by chance. Our results pose a challenge to most prevailing theories aimed at explaining the evolution of the mutation rate, underscoring the importance of obtaining additional mutation rate estimates in more genotypes, for more types of mutation, in more species, in order to improve our future understanding of mutation rates, their variation, and their evolution.

Engines of change: Transposable element mutation rates are high and variable within Daphnia magna.

Transposable elements (TEs) represent a major portion of most eukaryotic genomes, yet little is known about their mutation rates or how their activity is shaped by other evolutionary forces. Here, we compare short- and long-term patterns of genome-wide mutation accumulation (MA) of TEs among 9 genotypes from three populations of Daphnia magna from across a latitudinal gradient. While the overall proportion of the genome comprised of TEs is highly similar among genotypes from Finland, Germany, and Israel, populations are distinguishable based on patterns of insertion site polymorphism. Our direct rate estimates indicate TE movement is highly variable (net rates ranging from -11.98 to 12.79 x 10-5 per copy per generation among genotypes), differing both among populations and TE families. Although gains outnumber losses when selection is minimized, both types of events appear to be highly deleterious based on their low frequency in control lines where propagation is not limited to random, single-progeny descent. With rate estimates 4 orders of magnitude higher than base substitutions, TEs clearly represent a highly mutagenic force in the genome. Quantifying patterns of intra- and interspecific variation in TE mobility with and without selection provides insight into a powerful mechanism generating genetic variation in the genome.

Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits.

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.

Genome-wide identification of accessible chromatin regions in bumblebee by ATAC-seq.

Bumblebees (Hymenoptera: Apidae) are important pollinating insects that play pivotal roles in crop production and natural ecosystem services. Although protein-coding genes in bumblebees have been extensively annotated, regulatory sequences of the genome, such as promoters and enhancers, have been poorly annotated. To achieve a comprehensive profile of accessible chromatin regions and provide clues for all possible regulatory elements in the bumblebee genome, we performed ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) on Bombus terrestris samples derived from four developmental stages: egg, larva, pupa, and adult, respectively. The ATAC-seq reads were mapped to the B. terrestris reference genome, and its accessible chromatin regions were identified and characterized using bioinformatic methods. We identified 36,390 chromatin accessible regions in total, including both shared and stage-specific chromatin accessible signals. Our study will provide an important resource, not only for uncovering regulatory elements in the bumblebee genome, but also for expanding our understanding of bumblebee biology throughout development.

High and Highly Variable Spontaneous Mutation Rates in Daphnia.

The rate and spectrum of spontaneous mutations are critical parameters in basic and applied biology because they dictate the pace and character of genetic variation introduced into populations, which is a prerequisite for evolution. We use a mutation-accumulation approach to estimate mutation parameters from whole-genome sequence data from multiple genotypes from multiple populations of Daphnia magna, an ecological and evolutionary model system. We report extremely high base substitution mutation rates (µ-n,bs = 8.96 × 10-9/bp/generation [95% CI: 6.66-11.97 × 10-9/bp/generation] in the nuclear genome and µ-m,bs = 8.7 × 10-7/bp/generation [95% CI: 4.40-15.12 × 10-7/bp/generation] in the mtDNA), the highest of any eukaryote examined using this approach. Levels of intraspecific variation based on the range of estimates from the nine genotypes collected from three populations (Finland, Germany, and Israel) span 1 and 3 orders of magnitude, respectively, resulting in up to a ∼300-fold difference in rates among genomic partitions within the same lineage. In contrast, mutation spectra exhibit very consistent patterns across genotypes and populations, suggesting the mechanisms underlying the mutational process may be similar, even when the rates at which they occur differ. We discuss the implications of high levels of intraspecific variation in rates, the importance of estimating gene conversion rates using a mutation-accumulation approach, and the interacting factors influencing the evolution of mutation parameters. Our findings deepen our knowledge about mutation and provide both challenges to and support for current theories aimed at explaining the evolution of the mutation rate, as a trait, across taxa.

Variation in the Microbiota Associated with Daphnia magna Across Genotypes, Populations, and Temperature.

Studies of how the microbiome varies among individuals, populations, and abiotic conditions are critical for understanding this key component of an organism's biology and ecology. In the case of Daphnia, aquatic microcrustaceans widely used in population/community ecology and environmental science studies, understanding factors that influence microbiome shifts among individuals is useful for both basic and applied research contexts. In this study, we assess differences in the microbiome among genotypes of D. magna collected from three regions along a large latitudinal gradient (Finland, Germany, and Israel). After being reared in the lab for many years, we sought to characterize any differences in genotype- or population-specific microbial communities, and to assess whether the microbiota varied among temperatures. Our study is similar to a recent comparison of the microbial communities among D. magna genotypes raised in different temperatures published by Sullam et al. (Microb Ecol 76(2):506-517, 2017), and as such represents one of the first examples of a reproducible result in microbiome research. Like the previous study, we find evidence for a strong effect of temperature on the microbiome of D. magna, although across a much smaller temperature range representing potential near-future climates. In addition, we find evidence that the microbiomes of D. magna genotypes from different regions are distinct, even years after being brought into the laboratory. Finally, our results highlight a potentially common finding in the expanding area of microbiome research-differences among treatments are not necessarily observed in the most abundant taxonomic groups. This highlights the importance of considering sampling scheme and depth of coverage when characterizing the microbiome, as different experimental designs can significantly impact taxon-specific results, even when large-scale effects are reproduced.

Disentangling the intertwined roles of mutation, selection and drift in the mitochondrial genome.

Understanding and quantifying the rates of change in the mitochondrial genome is a major component of many areas of biological inquiry, from phylogenetics to human health. A critical parameter in understanding rates of change is estimating the mitochondrial mutation rate (mtDNA MR). Although the first direct estimates of mtDNA MRs were reported almost 20 years ago, the number of estimates has not grown markedly since that time. This is largely owing to the challenges associated with time- and labour-intensive mutation accumulation (MA) experiments. But even MA experiments do not solve a major problem with estimating mtDNA MRs-the challenge of disentangling the role of mutation from other evolutionary forces acting within the cell. Now that it is widely understood that any newly generated mutant allele in the mitochondria will initially be at very low frequency (1/N, where N is the number of mtDNA molecules in the cell), the importance of understanding the effective population size (Ne) of the mtDNA and the size of genetic bottlenecks during gametogenesis and development has come into the spotlight. In addition to these factors regulating the role of genetic drift, advances in our understanding of mitochondrial replication and turnover allow us to more easily envision how natural selection within the cell might favour or purge mutations in multi-copy organellar genomes. Here, we review the unique features of the mitochondrial genome that pose a challenge for accurate MR estimation and discuss ways to overcome those challenges. Estimates of mtDNA MRs remain one of the most widely used parameters in biology, thus accurate quantification and a deeper understanding of how and why they may vary within and between individuals, populations and species is an important goal. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Draft Genome of Busseola fusca, the Maize Stalk Borer, a Major Crop Pest in Sub-Saharan Africa.

The maize stalk borer, Busseola fusca, is an important Lepidopteran pest of cereal crops in Central, East, and Southern Africa. Crop losses due to B. fusca feeding activity vary by region, but can result in total crop loss in areas with high levels of infestation. Genomic resources provide critical insight into the biology of pest species and can allow for the development of effective management tools and strategies to mitigate their impact on agriculture. To this end, we sequenced, assembled, and annotated the genome of B. fusca. The total assembled genome size was 492.9 Mb with 19,417 annotated protein-coding genes. Using a comparative approach, we identified a putative expansion in the Chorion gene family, which is involved in the formation of the egg shell structure. Our analysis revealed high repeat content within the B. fusca genome, with LTR sequences comprising the majority of the repetitive sequence. We hope genomic resources will provide a foundation for future work aimed at developing an integrated pest management strategy to reduce B. fusca's impact on food security.

Intraspecific Variation in Microsatellite Mutation Profiles in Daphnia magna.

Microsatellite loci (tandem repeats of short nucleotide motifs) are highly abundant in eukaryotic genomes and often used as genetic markers because they can exhibit variation both within and between populations. Although widely recognized for their mutability and utility, the mutation rates of microsatellites have only been empirically estimated in a few species, and have rarely been compared across genotypes and populations within a species. Here, we investigate the dynamics of microsatellite mutation over long- and short-time periods by quantifying the starting abundance and mutation rates for microsatellites for six different genotypes of Daphnia magna, an aquatic microcrustacean, collected from three populations (Finland, Germany, and Israel). Using whole-genome sequences of these six starting genotypes, descendent mutation accumulation (MA) lines, and large population controls (non-MA lines), we find each genotype exhibits a distinctive initial microsatellite profile which clusters according to the population-of-origin. During the period of MA, we observe motif-specific, highly variable, and rapid microsatellite mutation rates across genotypes of D. magna, the average of which is order of magnitude greater than the recently reported rate observed in a single genotype of the congener, Daphnia pulex. In our experiment, genotypes with more microsatellites starting out exhibit greater losses and those with fewer microsatellites starting out exhibit greater gains-a context-dependent mutation bias that has not been reported previously. We discuss how genotype-specific mutation rates and spectra, in conjunction with evolutionary forces, can shape both the differential accumulation of repeat content in the genome and the evolution of mutation rates.

Tandem Repeats Contribute to Coding Sequence Variation in Bumblebees (Hymenoptera: Apidae).

Tandem repeats (TRs) are highly dynamic regions of the genome. Mutations at these loci represent a significant source of genetic variation and can facilitate rapid adaptation. Bumblebees are important pollinating insects occupying a wide range of habitats. However, to date, molecular mechanisms underlying the potential adaptation of bumblebees to diverse habitats are largely unknown. In the present study, we investigate how TRs contribute to genetic variation in bumblebees, thus potentially facilitating adaptation. We identified 26,595 TRs from the assembled 18 chromosome sequences of the buff-tailed bumblebee (Bombus terrestris), 66.7% of which reside in genic regions. We also compared TRs found in B. terrestris with those present in the assembled genome sequence of a congener, B. impatiens. We found that a total of 1,137 TRs were variable in length between the two sequenced bumblebee species, and further analysis reveals that 101 of them are located within coding regions. These 101 TRs are responsible for coding sequence variation and correspond to protein sequence length variation between the two bumblebee species. The variability of identified TRs in coding regions between bumblebees was confirmed by PCR amplification of a subset of loci. Functional classification of bumblebee genes where coding sequences include variable-length TRs suggests that a majority of genes (87%) that could be assigned to a protein class are related to transcriptional regulation. Our results show that TRs contribute to coding sequence variation in bumblebees, and thus may facilitate the adaptation of bumblebees through diversifying proteins involved in controlling gene expression.

Hierarchical Assessment of Mutation Properties in Daphnia magna.

Understanding the context-dependence of spontaneous mutations is crucial to predicting evolutionary trajectories. In this experiment, the impact of genetic background and trait-type on mutational susceptibility was investigated. Mutant and non-mutant lines of six unique genotypes from two populations of Daphnia magna were phenotypically assayed using a common-garden experiment. Morphological, life-history, and behavioral traits were measured and estimates of the mutation parameters were generated. The mutation parameters varied between the populations and among genotypes, suggesting differential susceptibility to mutation depending upon genomic background. Traits also varied in their susceptibility to mutation with behavioral traits evolving more rapidly than life-history and morphological traits. These results may reflect the unique selection histories of these populations.

Evolutionary Conflict between Mobile DNA and Host Genomes.

The proportion of eukaryotic genomes composed of active or formerly active mobile elements (MEs) is known to vary widely across lineages, but the explanations for why remain largely unknown. Given that ME activity, like other forms of mutation, is thought to be (on average) slightly deleterious in terms of phenotypic effects, understanding the widespread proliferation of MEs in host genomes requires an evolutionary framework. To better develop such a framework, we review the spectrum of resolutions to the genetic conflict between MEs and their hosts: inactivation of MEs due to mutation accumulation, negative selection (or lack thereof) against hosts with high ME loads, silencing of MEs (by hosts or MEs), ME domestication by their hosts, and the horizontal transfer of MEs to new hosts. We also highlight ecological and evolutionary theory from which ME researchers might borrow in order to explain large-scale patterns of ME dynamics across systems. We hope that a synthesis of the surprisingly significant role played by MEs in the genome, as well as the spectrum of resolutions, applicable theory, and recent discoveries, will have two outcomes for future researchers: better parsing of known variation in ME proliferation patterns across genomes and the development of testable models and predictions regarding the evolutionary trajectory of MEs based on a combination of theory, the comparative method, experimental evolution, and empirical observations.

Genome-Wide Estimates of Transposable Element Insertion and Deletion Rates in Drosophila Melanogaster.

Knowing the rate at which transposable elements (TEs) insert and delete is critical for understanding their role in genome evolution. We estimated spontaneous rates of insertion and deletion for all known, active TE superfamilies present in a set of Drosophila melanogaster mutation-accumulation (MA) lines using whole genome sequence data. Our results demonstrate that TE insertions far outpace TE deletions in D. melanogaster. We found a significant effect of background genotype on TE activity, with higher rates of insertions in one MA line. We also found significant rate heterogeneity between the chromosomes, with both insertion and deletion rates elevated on the X relative to the autosomes. Further, we identified significant associations between TE activity and chromatin state, and tested for associations between TE activity and other features of the local genomic environment such as TE content, exon content, GC content, and recombination rate. Our results provide the most detailed assessment of TE mobility in any organism to date, and provide a useful benchmark for both addressing theoretical predictions of TE dynamics and for exploring large-scale patterns of TE movement in D. melanogaster and other species.

Understanding Ebola: the 2014 epidemic.

Near the end of 2013, an outbreak of Zaire ebolavirus (EBOV) began in Guinea, subsequently spreading to neighboring Liberia and Sierra Leone. As this epidemic grew, important public health questions emerged about how and why this outbreak was so different from previous episodes. This review provides a synthetic synopsis of the 2014-15 outbreak, with the aim of understanding its unprecedented spread. We present a summary of the history of previous epidemics, describe the structure and genetics of the ebolavirus, and review our current understanding of viral vectors and the latest treatment practices. We conclude with an analysis of the public health challenges epidemic responders faced and some of the lessons that could be applied to future outbreaks of Ebola or other viruses.

High mutational rates of large-scale duplication and deletion in Daphnia pulex.

Knowledge of the genome-wide rate and spectrum of mutations is necessary to understand the origin of disease and the genetic variation driving all evolutionary processes. Here, we provide a genome-wide analysis of the rate and spectrum of mutations obtained in two Daphnia pulex genotypes via separate mutation-accumulation (MA) experiments. Unlike most MA studies that utilize haploid, homozygous, or self-fertilizing lines, D. pulex can be propagated ameiotically while maintaining a naturally heterozygous, diploid genome, allowing the capture of the full spectrum of genomic changes that arise in a heterozygous state. While base-substitution mutation rates are similar to those in other multicellular eukaryotes (about 4 × 10(-9) per site per generation), we find that the rates of large-scale (>100 kb) de novo copy-number variants (CNVs) are significantly elevated relative to those seen in previous MA studies. The heterozygosity maintained in this experiment allowed for estimates of gene-conversion processes. While most of the conversion tract lengths we report are similar to those generated by meiotic processes, we also find larger tract lengths that are indicative of mitotic processes. Comparison of MA lines to natural isolates reveals that a majority of large-scale CNVs in natural populations are removed by purifying selection. The mutations observed here share similarities with disease-causing, complex, large-scale CNVs, thereby demonstrating that MA studies in D. pulex serve as a system for studying the processes leading to such alterations.

Genetic differentiation within a widespread "supertramp" taxon: Molecular phylogenetics of the Louisiade White-eye (Zosterops griseotinctus).

"Supertramp" species are highly specialized overwater dispersers, and are useful taxa for investigating the influence of dispersal ability on speciation and diversification in island settings. The Louisiade White-eye (Zosterops griseotinctus) is a widespread avian supertramp endemic to Papua New Guinea's offshore islands. We used maximum likelihood and Bayesian inference to reconstruct phylogenetic relationships based on 2 mitochondrial and 1 nuclear loci (1813 bp total) from 88 individuals representing all 4 named subspecies and the full breadth of the species' range. We found significant geographic and population genetic structure, and support for a major clade containing the coral islets of the central Louisiade Archipelago and outlying Nissan Island. We found evidence of metapopulation structure and gene flow within the Louisiade Archipelago clade, and relatively high genetic distinctiveness of outlying island populations, including the population on volcanically-defaunated Long Island. We reject a hypothesis of panmixia within the Louisiade White-eye despite their long-range dispersal ability, and find evidence of selection against dispersal ability in populations on high-elevation islands where disturbance is rare. Our study represents a rare intraspecies phylogeny of an avian supertramp, and sheds light on patterns of evolution in highly vagile island species.