High-Quality Genome Assemblies Reveal Evolutionary Dynamics of Repetitive DNA and Structural Rearrangements in the Drosophila virilis Subgroup.

High-quality genome assemblies across a range of nontraditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. Here, we used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. We find that our contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. We also leverage a small panel of resequenced strains to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. We further find that a neo-X chromosome in Drosophila americana displays X-like levels of nucleotide diversity. We also found that unusual repetitive elements were responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to Drosophila novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by 3-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. Our results represent a major advance in our understanding of genome biology in this emerging model clade.

The implications of satellite DNA instability on cellular function and evolution.

Abundant tandemly repeated satellite DNA is present in most eukaryotic genomes. Previous limitations including a pervasive view that it was uninteresting junk DNA, combined with challenges in studying it, are starting to dissolve - and recent studies have found important functions for satellite DNAs. The observed rapid evolution and implied instability of satellite DNA now has important significance for their functions and maintenance within the genome. In this review, we discuss the processes that lead to satellite DNA copy number instability, and the importance of mechanisms to manage the potential negative effects of instability. Satellite DNA is vulnerable to challenges during replication and repair, since it forms difficult-to-process secondary structures and its homology within tandem arrays can result in various types of recombination. Satellite DNA instability may be managed by DNA or chromatin-binding proteins ensuring proper nuclear localization and repair, or by proteins that process aberrant structures that satellite DNAs tend to form. We also discuss the pattern of satellite DNA mutations from recent mutation accumulation (MA) studies that have tracked changes in satellite DNA for up to 1000 generations with minimal selection. Finally, we highlight examples of satellite evolution from studies that have characterized satellites across millions of years of Drosophila fruit fly evolution, and discuss possible ways that selection might act on the satellite DNA composition.

High quality genome assemblies reveal evolutionary dynamics of repetitive DNA and structural rearrangements in the Drosophila virilis sub-group.

High-quality genome assemblies across a range of non-traditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. Here we used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. We find that our contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. We also leverage a small panel of resequenced strains to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. We further find that a neo-X chromosome in D. americana displays X-like levels of nucleotide diversity. We also found that unusual repetitive elements were responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to D. novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by three-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. Our results represent a major advance in our understanding of genome biology in this emerging model clade.

Three recent sex chromosome-to-autosome fusions in a Drosophila virilis strain with high satellite DNA content.

The karyotype, or number and arrangement of chromosomes, has varying levels of stability across both evolution and disease. Karyotype changes often originate from DNA breaks near the centromeres of chromosomes, which generally contain long arrays of tandem repeats or satellite DNA. Drosophila virilis possesses among the highest relative satellite abundances of studied species, with almost half its genome composed of three related 7 bp satellites. We discovered a strain of D. virilis that we infer recently underwent three independent chromosome fusion events involving the X and Y chromosomes, in addition to one subsequent fission event. Here, we isolate and characterize the four different karyotypes we discovered in this strain which we believe demonstrates remarkable genome instability. We discovered that one of the substrains with an X-autosome fusion has an X-to-Y chromosome nondisjunction rate 20 × higher than the D. virilis reference strain (21% vs 1%). Finally, we found an overall higher rate of DNA breakage in the substrain with higher satellite DNA compared to a genetically similar substrain with less satellite DNA. This suggests that satellite DNA abundance may play a role in the risk of genome instability. Overall, we introduce a novel system consisting of a single strain with four different karyotypes, which we believe will be useful for future studies of genome instability, centromere function, and sex chromosome evolution.

Genome assembly, structural variants, and genetic differentiation between lake whitefish young species pairs (Coregonus sp.) with long and short reads.

Nascent pairs of ecologically differentiated species offer an opportunity to get a better glimpse at the genetic architecture of speciation. Of particular interest is our recent ability to consider a wider range of genomic variants, not only single-nucleotide polymorphisms (SNPs), thanks to long-read sequencing technology. We can now identify structural variants (SVs) such as insertions, deletions and other rearrangements, allowing further insights into the genetic architecture of speciation and how different types of variants are involved in species differentiation. Here, we investigated genomic patterns of differentiation between sympatric species pairs (Dwarf and Normal) belonging to the lake whitefish (Coregonus clupeaformis) species complex. We assembled the first reference genomes for both C. clupeaformis sp. Normal and C. clupeaformis sp. Dwarf, annotated the transposable elements and analysed the genomes in the light of related coregonid species. Next, we used a combination of long- and short-read sequencing to characterize SVs and genotype them at the population scale using genome-graph approaches, showing that SVs cover five times more of the genome than SNPs. We then integrated both SNPs and SVs to investigate the genetic architecture of species differentiation in two different lakes and highlighted an excess of shared outliers of differentiation. In particular, a large fraction of SVs differentiating the two species correspond to insertions or deletions of transposable elements (TEs), suggesting that TE accumulation may represent a key component of genetic divergence between the Dwarf and Normal species. Together, our results suggest that SVs may play an important role in speciation and that, by combining second- and third-generation sequencing, we now have the ability to integrate SVs into speciation genomics.

Copy number evolution in simple and complex tandem repeats across the C57BL/6 and C57BL/10 inbred mouse lines.

Simple sequence tandem repeats are among the most rapidly evolving compartments of the genome. Some repeat expansions are associated with mammalian disease or meiotic segregation distortion, yet the rates of copy number change across generations are not well known. Here, we use 14 distinct sublineages of the C57BL/6 and C57BL/10 inbred mouse strains, which have been evolving independently over about 300 generations, to estimate the rates of copy number changes in genome-wide tandem repeats. Rates of change varied across repeats and across lines. Notably, CAG, whose expansions in coding regions are associated with many neurological and genetic disorders, was highly stable in copy number, likely indicating stabilizing selection. Rates of change were positively correlated with copy number, but the direction and magnitude of changes varied across lines. Some mouse lines experienced consistent losses or gains across most simple repeats, but this did not correlate with copy number changes in complex repeats. Rates of copy number change were similar between simple repeats and the more abundant complex repeats after normalization by copy number. Finally, the Y-specific centromeric repeat had a fourfold higher rate of change than the homologous centromeric repeat on other chromosomes. Structural differences in satellite complexity, or restriction to the Y chromosome and elevated mutation rates of the male germline, may explain the higher rate of change. Overall, our work underscores the mutational fluidity of long tandem arrays of repeats, and the correlations and constraints between genome-wide tandem repeats, which suggest that turnover is not a completely neutral process.

RepeatModeler2 for automated genomic discovery of transposable element families.

The accelerating pace of genome sequencing throughout the tree of life is driving the need for improved unsupervised annotation of genome components such as transposable elements (TEs). Because the types and sequences of TEs are highly variable across species, automated TE discovery and annotation are challenging and time-consuming tasks. A critical first step is the de novo identification and accurate compilation of sequence models representing all of the unique TE families dispersed in the genome. Here we introduce RepeatModeler2, a pipeline that greatly facilitates this process. This program brings substantial improvements over the original version of RepeatModeler, one of the most widely used tools for TE discovery. In particular, this version incorporates a module for structural discovery of complete long terminal repeat (LTR) retroelements, which are widespread in eukaryotic genomes but recalcitrant to automated identification because of their size and sequence complexity. We benchmarked RepeatModeler2 on three model species with diverse TE landscapes and high-quality, manually curated TE libraries: Drosophila melanogaster (fruit fly), Danio rerio (zebrafish), and Oryza sativa (rice). In these three species, RepeatModeler2 identified approximately 3 times more consensus sequences matching with >95% sequence identity and sequence coverage to the manually curated sequences than the original RepeatModeler. As expected, the greatest improvement is for LTR retroelements. Thus, RepeatModeler2 represents a valuable addition to the genome annotation toolkit that will enhance the identification and study of TEs in eukaryotic genome sequences. RepeatModeler2 is available as source code or a containerized package under an open license (https://github.com/Dfam-consortium/RepeatModeler, http://www.repeatmasker.org/RepeatModeler/).

Evolutionary Dynamics of Abundant 7-bp Satellites in the Genome of Drosophila virilis.

The factors that drive the rapid changes in abundance of tandem arrays of highly repetitive sequences, known as satellite DNA, are not well understood. Drosophila virilis has one of the highest relative amounts of simple satellites of any organism that has been studied, with an estimated >40% of its genome composed of a few related 7-bp satellites. Here, we use D. virilis as a model to understand technical biases affecting satellite sequencing and the evolutionary processes that drive satellite composition. By analyzing sequencing data from Illumina, PacBio, and Nanopore platforms, we identify platform-specific biases and suggest best practices for accurate characterization of satellites by sequencing. We use comparative genomics and cytogenetics to demonstrate that the highly abundant AAACTAC satellite family arose from a related satellite in the branch leading to the virilis phylad 4.5-11 Ma before exploding in abundance in some species of the clade. The most abundant satellite is conserved in sequence and location in the pericentromeric region but has diverged widely in abundance among species, whereas the satellites nearest the centromere are rapidly turning over in sequence composition. By analyzing multiple strains of D. virilis, we saw that the abundances of two centromere-proximal satellites are anticorrelated along a geographical gradient, which we suggest could be caused by ongoing conflicts at the centromere. In conclusion, we illuminate several key attributes of satellite evolutionary dynamics that we hypothesize to be driven by processes including selection, meiotic drive, and constraints on satellite sequence and abundance.

Accelerated rates of large-scale mutations in the presence of copper and nickel.

Mutation rate variation has been under intense investigation for decades. Despite these efforts, little is known about the extent to which environmental stressors accelerate mutation rates and influence the genetic load of populations. Moreover, most studies on stressors have focused on unicellular organisms and point mutations rather than large-scale deletions and duplications (copy number variations [CNVs]). We estimated mutation rates in Daphnia pulex exposed to low levels of environmental stressors as well as the effect of selection on de novo mutations. We conducted a mutation accumulation (MA) experiment in which selection was minimized, coupled with an experiment in which a population was propagated under competitive conditions in a benign environment. After an average of 103 generations of MA propagation, we sequenced 60 genomes and found significantly accelerated rates of deletions and duplications in MA lines exposed to ecologically relevant concentrations of metals. Whereas control lines had gene deletion and duplication rates comparable to other multicellular eukaryotes (1.8 × 10-6 per gene per generation), the presence of nickel and copper increased these rates fourfold. The realized mutation rate under selection was reduced to 0.4× that of control MA lines, providing evidence that CNVs contribute to mutational load. Our CNV breakpoint analysis revealed that nonhomologous recombination associated with regions of DNA fragility is the primary source of CNVs, plausibly linking metal-induced DNA strand breaks with higher CNV rates. Our findings suggest that environmental stress, in particular multiple stressors, can have profound effects on large-scale mutation rates and mutational load of multicellular organisms.

Fitness and Genomic Consequences of Chronic Exposure to Low Levels of Copper and Nickel in Daphnia pulex Mutation Accumulation Lines.

In at least some unicellular organisms, mutation rates are temporarily raised upon exposure to environmental stress, potentially contributing to the evolutionary response to stress. Whether this is true for multicellular organisms, however, has received little attention. This study investigated the effects of chronic mild stress, in the form of low-level copper and nickel exposure, on mutational processes in Daphnia pulex using a combination of mutation accumulation, whole genome sequencing and life-history assays. After over 100 generations of mutation accumulation, we found no effects of metal exposure on the rates of single nucleotide mutations and of loss of heterozygosity events, the two mutation classes that occurred in sufficient numbers to allow statistical analysis. Similarly, rates of decline in fitness, as measured by intrinsic rate of population increase and of body size at first reproduction, were negligibly affected by metal exposure. We can reject the possibility that Daphnia were insufficiently stressed to invoke genetic responses as we have previously shown rates of large-scale deletions and duplications are elevated under metal exposure in this experiment. Overall, the mutation accumulation lines did not significantly depart from initial values for phenotypic traits measured, indicating the lineage used was broadly mutationally robust. Taken together, these results indicate that the mutagenic effects of chronic low-level exposure to these metals are restricted to certain mutation classes and that fitness consequences are likely minor and therefore unlikely to be relevant in determining the evolutionary responses of populations exposed to these stressors.

Rates and Patterns of Mutation in Tandem Repetitive DNA in Six Independent Lineages of Chlamydomonas reinhardtii.

The mutational patterns of large tandem arrays of short sequence repeats remain largely unknown, despite observations of their high levels of variation in sequence and genomic abundance within and between species. Many factors can influence the dynamics of tandem repeat evolution; however, their evolution has only been examined over a limited phylogenetic sample of taxa. Here, we use publicly available whole-genome sequencing data of 85 haploid mutation accumulation lines derived from six geographically diverse Chlamydomonas reinhardtii isolates to investigate genome-wide mutation rates and patterns in tandem repeats in this species. We find that tandem repeat composition differs among ancestral strains, both in genome-wide abundance and presence/absence of individual repeats. Estimated mutation rates (repeat copy number expansion and contraction) were high, averaging 4.3×10-4 per generation per single unit copy. Although orders of magnitude higher than other types of mutation previously reported in C. reinhardtii, these tandem repeat mutation rates were one order of magnitude lower than what has recently been found in Daphnia pulex, even after correcting for lower overall genome-wide satellite abundance in C. reinhardtii. Most high-abundance repeats were related to others by a single mutational step. Correlations of repeat copy number changes within genomes revealed clusters of closely related repeats that were strongly correlated positively or negatively, and similar patterns of correlation arose independently in two different mutation accumulation experiments. Together, these results paint a dynamic picture of tandem repeat evolution in this unicellular alga.

Selection Constrains High Rates of Tandem Repetitive DNA Mutation in Daphnia pulex.

A long-standing evolutionary puzzle is that all eukaryotic genomes contain large amounts of tandemly-repeated DNA whose sequence motifs and abundance vary greatly among even closely related species. To elucidate the evolutionary forces governing tandem repeat dynamics, quantification of the rates and patterns of mutations in repeat copy number and tests of its selective neutrality are necessary. Here, we used whole-genome sequences of 28 mutation accumulation (MA) lines of Daphnia pulex, in addition to six isolates from a non-MA population originating from the same progenitor, to both estimate mutation rates of abundances of repeat sequences and evaluate the selective regime acting upon them. We found that mutation rates of individual repeats were both high and highly variable, ranging from additions/deletions of 0.29-105 copies per generation (reflecting changes of 0.12-0.80% per generation). Our results also provide evidence that new repeat sequences are often formed from existing ones. The non-MA population isolates showed a signal of either purifying or stabilizing selection, with 33% lower variation in repeat copy number on average than the MA lines, although the level of selective constraint was not evenly distributed across all repeats. The changes between many pairs of repeats were correlated, and the pattern of correlations was significantly different between the MA lines and the non-MA population. Our study demonstrates that tandem repeats can experience extremely rapid evolution in copy number, which can lead to high levels of divergence in genome-wide repeat composition between closely related species.

Spontaneous Mutation Accumulation in Daphnia pulex in Selection-Free vs. Competitive Environments.

Understanding the rates, spectra, and fitness effects of spontaneous mutations is fundamental to answering key questions in evolution, molecular biology, disease genetics, and conservation biology. To estimate mutation rates and evaluate the effect of selection on new mutations, we propagated mutation accumulation (MA) lines of Daphnia pulex for more than 82 generations and maintained a non-MA population under conditions where selection could act. Both experiments were started with the same obligate asexual progenitor clone. By sequencing 30 genomes and implementing a series of validation steps that informed the bioinformatic analyses, we identified a total of 477 single nucleotide mutations (SNMs) in the MA lines, corresponding to a mutation rate of 2.30 × 10-9 (95% CI 1.90-2.70 × 10-9) per nucleotide per generation. The high overall rate of loss of heterozygosity (LOH) of 4.82 × 10-5 per site per generation was due to a large ameiotic recombination event spanning an entire arm of a chromosome (∼6 Mb) and several hemizygous deletion events spanning ∼2 kb each. In the non-MA population, we found significantly fewer mutations than expected based on the rate derived from the MA experiment, indicating purifying selection was likely acting to remove new deleterious mutations. We observed a surprisingly high level of genetic variability in the non-MA population, which we propose to be driven by balancing selection. Our findings suggest that both positive and negative selection on new mutations is powerful and effective in a strictly clonal population.

Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods.

Metabarcoding has the potential to become a rapid, sensitive, and effective approach for identifying species in complex environmental samples. Accurate molecular identification of species depends on the ability to generate operational taxonomic units (OTUs) that correspond to biological species. Due to the sometimes enormous estimates of biodiversity using this method, there is a great need to test the efficacy of data analysis methods used to derive OTUs. Here, we evaluate the performance of various methods for clustering length variable 18S amplicons from complex samples into OTUs using a mock community and a natural community of zooplankton species. We compare analytic procedures consisting of a combination of (1) stringent and relaxed data filtering, (2) singleton sequences included and removed, (3) three commonly used clustering algorithms (mothur, UCLUST, and UPARSE), and (4) three methods of treating alignment gaps when calculating sequence divergence. Depending on the combination of methods used, the number of OTUs varied by nearly two orders of magnitude for the mock community (60-5068 OTUs) and three orders of magnitude for the natural community (22-22191 OTUs). The use of relaxed filtering and the inclusion of singletons greatly inflated OTU numbers without increasing the ability to recover species. Our results also suggest that the method used to treat gaps when calculating sequence divergence can have a great impact on the number of OTUs. Our findings are particularly relevant to studies that cover taxonomically diverse species and employ markers such as rRNA genes in which length variation is extensive.