Genome assemblies of the simultaneously hermaphroditic flatworms Macrostomum cliftonense and Macrostomum hystrix.

The free-living, simultaneously hermaphroditic flatworms of the genus Macrostomum are increasingly used as model systems in various contexts. In particular, Macrostomum lignano, the only species of this group with a published genome assembly, has emerged as a model for the study of regeneration, reproduction, and stem-cell function. However, challenges have emerged due to M. lignano being a hidden polyploid, having recently undergone whole-genome duplication and chromosome fusion events. This complex genome architecture presents a significant roadblock to the application of many modern genetic tools. Hence, additional genomic resources for this genus are needed. Here, we present such resources for Macrostomum cliftonense and Macrostomum hystrix, which represent the contrasting mating behaviors of reciprocal copulation and hypodermic insemination found in the genus. We use a combination of PacBio long-read sequencing and Illumina shot-gun sequencing, along with several RNA-Seq data sets, to assemble and annotate highly contiguous genomes for both species. The assemblies span ∼227 and ∼220 Mb and are represented by 399 and 42 contigs for M. cliftonense and M. hystrix, respectively. Furthermore, high BUSCO completeness (∼84-85%), low BUSCO duplication rates (8.3-6.2%), and low k-mer multiplicity indicate that these assemblies do not suffer from the same assembly ambiguities of the M. lignano genome assembly, which can be attributed to the complex karyology of this species. We also show that these resources, in combination with the prior resources from M. lignano, offer an excellent foundation for comparative genomic research in this group of organisms.

Y-Linked Copy Number Polymorphism of Target of Rapamycin Is Associated with Sexual Size Dimorphism in Seed Beetles.

The Y chromosome is theorized to facilitate evolution of sexual dimorphism by accumulating sexually antagonistic loci, but empirical support is scarce. Due to the lack of recombination, Y chromosomes are prone to degenerative processes, which poses a constraint on their adaptive potential. Yet, in the seed beetle, Callosobruchus maculatus segregating Y linked variation affects male body size and thereby sexual size dimorphism (SSD). Here, we assemble C. maculatus sex chromosome sequences and identify molecular differences associated with Y-linked SSD variation. The assembled Y chromosome is largely euchromatic and contains over 400 genes, many of which are ampliconic with a mixed autosomal and X chromosome ancestry. Functional annotation suggests that the Y chromosome plays important roles in males beyond primary reproductive functions. Crucially, we find that, besides an autosomal copy of the gene target of rapamycin (TOR), males carry an additional TOR copy on the Y chromosome. TOR is a conserved regulator of growth across taxa, and our results suggest that a Y-linked TOR provides a male specific opportunity to alter body size. A comparison of Y haplotypes associated with male size difference uncovers a copy number variation for TOR, where the haplotype associated with decreased male size, and thereby increased sexual dimorphism, has two additional TOR copies. This suggests that sexual conflict over growth has been mitigated by autosome to Y translocation of TOR followed by gene duplications. Our results reveal that despite of suppressed recombination, the Y chromosome can harbor adaptive potential as a male-limited supergene.

Experimental Life History Evolution Results in Sex-specific Evolution of Gene Expression in Seed Beetles.

The patterns of reproductive timing and senescence vary within and across species owing to differences in reproductive strategies, but our understanding of the molecular underpinnings of such variation is incomplete. This is perhaps particularly true for sex differences. We investigated the evolution of sex-specific gene expression associated with life history divergence in replicated populations of the seed beetle Acanthoscelides obtectus, experimentally evolving under (E)arly or (L)ate life reproduction for >200 generations which has resulted in strongly divergent life histories. We detected 1,646 genes that were differentially expressed in E and L lines, consistent with a highly polygenic basis of life history evolution. Only 30% of differentially expressed genes were similarly affected in males and females. The evolution of long life was associated with significantly reduced sex differences in expression, especially in non-reproductive tissues. The expression differences were overall more pronounced in females, in accordance with their greater phenotypic divergence in lifespan. Functional enrichment analysis revealed differences between E and L beetles in gene categories previously implicated in aging, such as mitochondrial function and defense response. The results show that divergent life history evolution can be associated with profound changes in gene expression that alter the transcriptome in a sex-specific way, highlighting the importance of understanding the mechanisms of aging in each sex.

Mating strategy predicts gene presence/absence patterns in a genus of simultaneously hermaphroditic flatworms.

Gene repertoire turnover is a characteristic of genome evolution. However, we lack well-replicated analyses of presence/absence patterns associated with different selection contexts. Here, we study ∼100 transcriptome assemblies across Macrostomum, a genus of simultaneously hermaphroditic flatworms exhibiting multiple convergent shifts in mating strategy and associated reproductive morphologies. Many species mate reciprocally, with partners donating and receiving sperm at the same time. Other species convergently evolved to mate by hypodermic injection of sperm into the partner. We find that for orthologous transcripts annotated as expressed in the body region containing the testes, sequences from hypodermically inseminating species diverge more rapidly from the model species, Macrostomum lignano, and have a lower probability of being observed in other species. For other annotation categories, simpler models with a constant rate of similarity decay with increasing genetic distance from M. lignano match the observed patterns well. Thus, faster rates of sequence evolution for hypodermically inseminating species in testis-region genes result in higher rates of homology detection failure, yielding a signal of rapid evolution in sequence presence/absence patterns. Our results highlight the utility of considering appropriate null models for unobserved genes, as well as associating patterns of gene presence/absence with replicated evolutionary events in a phylogenetic context.

The genetic basis and adult reproductive consequences of developmental thermal plasticity.

Increasing temperature and thermal variability generate profound selection on populations. Given the fast rate of environmental change, understanding the role of plasticity and genetic adaptation in response to increasing temperatures is critical. This may be especially true for thermal effects on reproductive traits in which thermal fertility limits at high temperatures may be lower than for survival traits. Consequences of changing environments during development on adult phenotypes may be particularly problematic for core traits such as reproduction that begin early in development. Here we examine the consequences of developmental thermal plasticity on subsequent adult reproductive traits and its genetic basis. We used a panel of Drosophila melanogaster (the Drosophila Genetic Reference Panel; DGRP) in which male fertility performance was previously defined as either showing relatively little (status = 'high'-performing lines) or substantial ('low'-performing lines) decline when exposed to increasing developmental temperatures. We used a thermal reaction norm approach to quantify variation in the consequences of developmental thermal plasticity on multiple adult reproductive traits, including sex-specific responses, and to identify candidate genes underlying such variation. Developmental thermal stress impacted the means and thermal reaction norms of all reproductive traits except offspring sex ratio. Mating success declined as temperature increased with no difference between high and low lines, whereas increasing temperature resulted in declines for both male and female fertility and productivity but depended on line status. Fertility and offspring number were positively correlated within and between the sexes across lines, but males were more affected than females. We identified 933 SNPs with significant evolved genetic differentiation between high and low lines. In all, 54 of these lie within genomic windows of overall high differentiation, have significant effects of genotype on the male thermal reaction norm for productivity and are associated with 16 genes enriched for phenotypes affecting reproduction, stress responses and autophagy in Drosophila and other organisms. Our results illustrate considerable plasticity in male thermal limits on several reproductive traits following development at high temperature, and we identify differentiated loci with relevant phenotypic effects that may contribute to this population variation. While our work is on a single population, phenotypic results align with an increasing number of studies demonstrating the potential for stronger selection of thermal stress on reproductive traits, particularly in males. Such large fitness costs may have both short- and long-term consequences for the evolution of populations in response to a warming world.

Large-scale phylogenomics of the genus Macrostomum (Platyhelminthes) reveals cryptic diversity and novel sexual traits.

Free-living flatworms of the genus Macrostomum are small and transparent animals, representing attractive study organisms for a broad range of topics in evolutionary, developmental, and molecular biology. The genus includes the model organism M. lignano for which extensive molecular resources are available, and recently there is a growing interest in extending work to additional species in the genus. These endeavours are currently hindered because, even though >200 Macrostomum species have been taxonomically described, molecular phylogenetic information and geographic sampling remain limited. We report on a global sampling campaign aimed at increasing taxon sampling and geographic representation of the genus. Specifically, we use extensive transcriptome and single-locus data to generate phylogenomic hypotheses including 145 species. Across different phylogenetic methods and alignments used, we identify several consistent clades, while their exact grouping is less clear, possibly due to a radiation early in Macrostomum evolution. Moreover, we uncover a large undescribed diversity, with 94 of the studied species likely being new to science, and we identify multiple novel morphological traits. Furthermore, we identify cryptic speciation in a taxonomically challenging assemblage of species, suggesting that the use of molecular markers is a prerequisite for future work, and we describe the distribution of putative synapomorphies and suggest taxonomic revisions based on our finding. Our large-scale phylogenomic dataset now provides a robust foundation for comparative analyses of morphological, behavioural and molecular evolution in this genus.

Faster Rates of Molecular Sequence Evolution in Reproduction-Related Genes and in Species with Hypodermic Sperm Morphologies.

Sexual selection drives the evolution of many striking behaviors and morphologies and should leave signatures of selection at loci underlying these phenotypes. However, although loci thought to be under sexual selection often evolve rapidly, few studies have contrasted rates of molecular sequence evolution at such loci across lineages with different sexual selection contexts. Furthermore, work has focused on separate sexed animals, neglecting alternative sexual systems. We investigate rates of molecular sequence evolution in hermaphroditic flatworms of the genus Macrostomum. Specifically, we compare species that exhibit contrasting sperm morphologies, strongly associated with multiple convergent shifts in the mating strategy, reflecting different sexual selection contexts. Species donating and receiving sperm in every mating have sperm with bristles, likely to prevent sperm removal. Meanwhile, species that hypodermically inject sperm lack bristles, potentially as an adaptation to the environment experienced by hypodermic sperm. Combining functional annotations from the model, Macrostomum lignano, with transcriptomes from 93 congeners, we find genus-wide faster sequence evolution in reproduction-related versus ubiquitously expressed genes, consistent with stronger sexual selection on the former. Additionally, species with hypodermic sperm morphologies had elevated molecular sequence evolution, regardless of a gene's functional annotation. These genome-wide patterns suggest reduced selection efficiency following shifts to hypodermic mating, possibly due to higher selfing rates in these species. Moreover, we find little evidence for convergent amino acid changes across species. Our work not only shows that reproduction-related genes evolve rapidly also in hermaphroditic animals, but also that well-replicated contrasts of different sexual selection contexts can reveal underappreciated genome-wide effects.

Experimental evolution supports signatures of sexual selection in genomic divergence.

Comparative genomics has contributed to the growing evidence that sexual selection is an important component of evolutionary divergence and speciation. Divergence by sexual selection is implicated in faster rates of divergence of the X chromosome and of genes thought to underlie sexually selected traits, including genes that are sex biased in expression. However, accurately inferring the relative importance of complex and interacting forms of natural selection, demography, and neutral processes that occurred in the evolutionary past is challenging. Experimental evolution provides an opportunity to apply controlled treatments for multiple generations and examine the consequent genomic divergence. Here, we altered sexual selection intensity, elevating sexual selection in polyandrous lines and eliminating it in monogamous lines, and examined patterns of allele frequency divergence in the genome of Drosophila pseudoobscura after more than 160 generations of experimental evolution. Divergence is not uniform across the genome but concentrated in "islands," many of which contain candidate genes implicated in mating behaviors and other sexually selected phenotypes. These are more often seen on the X chromosome, which also shows greater divergence in F ST than neutral expectations. There are characteristic signatures of selection seen in these regions, with lower diversity on the X chromosome than the autosomes, and differences in diversity on the autosomes between selection regimes. Reduced Tajima's D within some of the divergent regions may imply that selective sweeps have occurred, despite considerable recombination. These changes are associated with both differential gene expression between the lines and sex-biased gene expression within the lines. Our results are very similar to those thought to implicate sexual selection in divergence between species and natural populations, and hence provide experimental support for the likely role of sexual selection in driving such types of genetic divergence, but also illustrate how variable outcomes can be for different genomic regions.

A genome-wide investigation of adaptive signatures in protein-coding genes related to tool behaviour in New Caledonian and Hawaiian crows.

Very few animals habitually manufacture and use tools. It has been suggested that advanced tool behaviour co-evolves with a suite of behavioural, morphological and life history traits. In fact, there are indications for such an adaptive complex in tool-using crows (genus Corvus species). Here, we sequenced the genomes of two habitually tool-using and ten non-tool-using crow species to search for genomic signatures associated with a tool-using lifestyle. Using comparative genomic and population genetic approaches, we screened for signals of selection in protein-coding genes in the tool-using New Caledonian and Hawaiian crows. While we detected signals of recent selection in New Caledonian crows near genes associated with bill morphology, our data indicate that genetic changes in these two lineages are surprisingly subtle, with little evidence at present for convergence. We explore the biological explanations for these findings, such as the relative roles of gene regulation and protein-coding changes, as well as the possibility that statistical power to detect selection in recently diverged lineages may have been insufficient. Our study contributes to a growing body of literature aiming to decipher the genetic basis of recently evolved complex behaviour.

RNA-Seq of three free-living flatworm species suggests rapid evolution of reproduction-related genes.

BACKGROUND: The genus Macrostomum consists of small free-living flatworms and contains Macrostomum lignano, which has been used in investigations of ageing, stem cell biology, bioadhesion, karyology, and sexual selection in hermaphrodites. Two types of mating behaviour occur within this genus. Some species, including M. lignano, mate via reciprocal copulation, where, in a single mating, both partners insert their male copulatory organ into the female storage organ and simultaneously donate and receive sperm. Other species mate via hypodermic insemination, where worms use a needle-like copulatory organ to inject sperm into the tissue of the partner. These contrasting mating behaviours are associated with striking differences in sperm and copulatory organ morphology. Here we expand the genomic resources within the genus to representatives of both behaviour types and investigate whether genes vary in their rate of evolution depending on their putative function. RESULTS: We present de novo assembled transcriptomes of three Macrostomum species, namely M. hystrix, a close relative of M. lignano that mates via hypodermic insemination, M. spirale, a more distantly related species that mates via reciprocal copulation, and finally M. pusillum, which represents a clade that is only distantly related to the other three species and also mates via hypodermic insemination. We infer 23,764 sets of homologous genes and annotate them using experimental evidence from M. lignano. Across the genus, we identify 521 gene families with conserved patterns of differential expression between juvenile vs. adult worms and 185 gene families with a putative expression in the testes that are restricted to the two reciprocally mating species. Further, we show that homologs of putative reproduction-related genes have a higher protein divergence across the four species than genes lacking such annotations and that they are more difficult to identify across the four species, indicating that these genes evolve more rapidly, while genes involved in neoblast function are more conserved. CONCLUSIONS: This study improves the genus Macrostomum as a model system, by providing resources for the targeted investigation of gene function in a broad range of species. And we, for the first time, show that reproduction-related genes evolve at an accelerated rate in flatworms.

Competition for access to mates predicts female-specific ornamentation and male investment in relative testis size.

Sexually selected ornaments are highly variable and the factors that drive variation in ornament expression are not always clear. Rare instances of female-specific ornament evolution (such as in some dance fly species) are particularly puzzling. While some evidence suggests that such rare instances represent straightforward reversals of sexual selection intensity, the distinct nature of trade-offs between ornaments and offspring pose special constraints in females. To examine whether competition for access to mates generally favors heightened ornament expression, we built a phylogeny and conducted a comparative analysis of Empidinae dance fly taxa that display female-specific ornaments. We show that species with more female-biased operational sex ratios in lek-like mating swarms have greater female ornamentation, and in taxa with more ornate females, male relative testis investment is increased. These findings support the hypothesis that ornament diversity in dance flies depends on female receptivity to mates, which is associated with contests for nutritious nuptial gifts provided by males. Moreover, our results suggest that increases in female receptivity lead to higher levels of sperm competition among males. The incidence of both heightened premating sexual selection on females and postmating selection on males contradicts assertions that sex roles are straightforwardly reversed in dance flies.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Purifying Selection in Corvids Is Less Efficient on Islands.

Theory predicts that deleterious mutations accumulate more readily in small populations. As a consequence, mutation load is expected to be elevated in species where life-history strategies and geographic or historical contingencies reduce the number of reproducing individuals. Yet, few studies have empirically tested this prediction using genome-wide data in a comparative framework. We collected whole-genome sequencing data for 147 individuals across seven crow species (Corvus spp.). For each species, we estimated the distribution of fitness effects of deleterious mutations and compared it with proxies of the effective population size Ne. Island species with comparatively smaller geographic range sizes had a significantly increased mutation load. These results support the view that small populations have an elevated risk of mutational meltdown, which may contribute to the higher extinction rates observed in island species.

Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence.

The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are inter-related is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity.

Inter and Intraspecific Genomic Divergence in Drosophila montana Shows Evidence for Cold Adaptation.

The genomes of species that are ecological specialists will likely contain signatures of genomic adaptation to their niche. However, distinguishing genes related to ecological specialism from other sources of selection and more random changes is a challenge. Here, we describe the genome of Drosophila montana, which is the most extremely cold-adapted Drosophila species known. We use branch tests to identify genes showing accelerated divergence in contrasts between cold- and warm-adapted species and identify about 250 genes that show differences, possibly driven by a lower synonymous substitution rate in cold-adapted species. We also look for evidence of accelerated divergence between D. montana and D. virilis, a previously sequenced relative, but do not find strong evidence for divergent selection on coding sequence variation. Divergent genes are involved in a variety of functions, including cuticular and olfactory processes. Finally, we also resequenced three populations of D. montana from across its ecological and geographic range. Outlier loci were more likely to be found on the X chromosome and there was a greater than expected overlap between population outliers and those genes implicated in cold adaptation between Drosophila species, implying some continuity of selective process at these different evolutionary scales.

Identifying consistent allele frequency differences in studies of stratified populations.

With increasing application of pooled-sequencing approaches to population genomics robust methods are needed to accurately quantify allele frequency differences between populations. Identifying consistent differences across stratified populations can allow us to detect genomic regions under selection and that differ between populations with different histories or attributes. Current popular statistical tests are easily implemented in widely available software tools which make them simple for researchers to apply. However, there are potential problems with the way such tests are used, which means that underlying assumptions about the data are frequently violated.These problems are highlighted by simulation of simple but realistic population genetic models of neutral evolution and the performance of different tests are assessed. We present alternative tests (including Generalised Linear Models [GLMs] with quasibinomial error structure) with attractive properties for the analysis of allele frequency differences and re-analyse a published dataset.The simulations show that common statistical tests for consistent allele frequency differences perform poorly, with high false positive rates. Applying tests that do not confound heterogeneity and main effects significantly improves inference. Variation in sequencing coverage likely produces many false positives and re-scaling allele frequencies to counts out of a common value or an effective sample size reduces this effect.Many researchers are interested in identifying allele frequencies that vary consistently across replicates to identify loci underlying phenotypic responses to selection or natural variation in phenotypes. Popular methods that have been suggested for this task perform poorly in simulations. Overall, quasibinomial GLMs perform better and also have the attractive feature of allowing correction for multiple testing by standard procedures and are easily extended to other designs.

The Genome and Methylome of a Beetle with Complex Social Behavior, Nicrophorus vespilloides (Coleoptera: Silphidae).

Testing for conserved and novel mechanisms underlying phenotypic evolution requires a diversity of genomes available for comparison spanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily with eusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from a wider range of taxa that also vary in their levels of sociality. Here, we present the assembled and annotated genome of the subsocial beetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used this genome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene models more strongly than phylogeny? We found that the overlap in gene models was similar between N. vespilloides and all other insect groups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, does N. vespilloides have DNA methylation? We found strong evidence for an active DNA methylation system. The distribution of methylation was similar to other insects with exons having the most methylated CpGs. Methylation status appears highly conserved; 85% of the methylated genes in N. vespilloides are also methylated in the hymentopteran Nasonia vitripennis. The addition of this genome adds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questions about the potential role of methylation in social behavior.

Assessing Recent Selection and Functionality at Long Noncoding RNA Loci in the Mouse Genome.

Long noncoding RNAs (lncRNAs) are one of the most intensively studied groups of noncoding elements. Debate continues over what proportion of lncRNAs are functional or merely represent transcriptional noise. Although characterization of individual lncRNAs has identified approximately 200 functional loci across the Eukarya, general surveys have found only modest or no evidence of long-term evolutionary conservation. Although this lack of conservation suggests that most lncRNAs are nonfunctional, the possibility remains that some represent recent evolutionary innovations. We examine recent selection pressures acting on lncRNAs in mouse populations. We compare patterns of within-species nucleotide variation at approximately 10,000 lncRNA loci in a cohort of the wild house mouse, Mus musculus castaneus, with between-species nucleotide divergence from the rat (Rattus norvegicus). Loci under selective constraint are expected to show reduced nucleotide diversity and divergence. We find limited evidence of sequence conservation compared with putatively neutrally evolving ancestral repeats (ARs). Comparisons of sequence diversity and divergence between ARs, protein-coding (PC) exons and lncRNAs, and the associated flanking regions, show weak, but significantly lower levels of sequence diversity and divergence at lncRNAs compared with ARs. lncRNAs conserved deep in the vertebrate phylogeny show lower within-species sequence diversity than lncRNAs in general. A set of 74 functionally characterized lncRNAs show levels of diversity and divergence comparable to PC exons, suggesting that these lncRNAs are under substantial selective constraints. Our results suggest that, in mouse populations, most lncRNA loci evolve at rates similar to ARs, whereas older lncRNAs tend to show signals of selection similar to PC genes.