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De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterize a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioinformatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit moderately higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic random sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system than random expectation, given their higher solubility.

Vertebrate blood coagulation is controlled by a cascade containing more than 20 proteins. The cascade proteins are found in the blood in their zymogen forms and when the cascade is triggered by tissue damage, zymogens are activated and in turn activate their downstream proteins by serine protease activity. In this study, we examined proteomes of 21 chordates, of which 18 are vertebrates, to reveal the modular evolution of the blood coagulation cascade. Additionally, two Arthropoda species were used to compare domain arrangements of the proteins belonging to the hemolymph clotting and the blood coagulation cascades. Within the vertebrate coagulation protein set, almost half of the studied proteins are shared with jawless vertebrates. Domain similarity analyses revealed that there are multiple possible evolutionary trajectories for each coagulation protein. During the evolution of higher vertebrate clades, gene and genome duplications led to the formation of other coagulation cascade proteins.

New protein coding genes can emerge from genomic regions that previously did not contain any genes, via a process called de novo gene emergence. To synthesize a protein, DNA must be transcribed as well as translated. Both processes need certain DNA sequence features. Stable transcription requires promoters and a polyadenylation signal, while translation requires at least an open reading frame. We develop mathematical models based on mutation probabilities, and the assumption of neutral evolution, to find out how quickly genes emerge and are lost. We also investigate the effect of the order by which DNA features evolve, and if sequence composition is biased by mutation rate. We rationalize how genes are lost much more rapidly than they emerge, and how they preferentially arise in regions that are already transcribed. Our study not only answers some fundamental questions on the topic of de novo emergence but also provides a modeling framework for future studies.

(1) Unravelling the molecular basis underlying major evolutionary transitions can shed light on how complex phenotypes arise. The evolution of eusociality, a major evolutionary transition, has been demonstrated to be accompanied by enhanced gene regulation. Numerous pieces of evidence suggest the major impact of transposon insertion on gene regulation and its role in adaptive evolution. Transposons have been shown to be play a role in gene duplication involved in the eusocial transition in termites. However, evidence of the molecular basis underlying the eusocial transition in Blattodea remains scarce. Could transposons have facilitated the eusocial transition in termites through shifts of gene expression? (2) Using available cockroach and termite genomes and transcriptomes, we investigated if transposons insert more frequently in genes with differential expression in queens and workers and if those genes could be linked to specific functions essential for eusocial transition. (3) The insertion rate of transposons differs among differentially expressed genes and displays opposite trends between termites and cockroaches. The functions of termite transposon-rich queen- and worker-biased genes are related to reproduction and ageing and behaviour and gene expression, respectively. (4) Our study provides further evidence on the role of transposons in the evolution of eusociality, potentially through shifts in gene expression.

Transposable elements (TEs) are mobile genetic sequences, which can cause the accumulation of genomic damage in the lifetime of an organism. The regulation of TEs, for instance via the piRNA-pathway, is an important mechanism to protect the integrity of genomes, especially in the germ-line where mutations can be transmitted to offspring. In eusocial insects, soma and germ-line are divided among worker and reproductive castes, so one may expect caste-specific differences in TE regulation to exist. To test this, we compared whole-genome levels of repeat element transcription in the fat body of female workers, kings and five different queen stages of the higher termite, Macrotermes natalensis. In this species, queens can live over 20 years, maintaining near maximum reproductive output, while sterile workers only live weeks. We found a strong, positive correlation between TE expression and the expression of neighbouring genes in all castes. However, we found substantially higher TE activity in workers than in reproductives. Furthermore, TE expression did not increase with age in queens, despite a sevenfold increase in overall gene expression, due to a significant upregulation of the piRNA-pathway in 20-year-old queens. Our results suggest a caste- and age-specific regulation of the piRNA-pathway has evolved in higher termites that is analogous to germ-line-specific activity in solitary organisms. In the fat body of these termite queens, an important metabolic tissue for maintaining their extreme longevity and reproductive output, an efficient regulation of TEs likely protects genome integrity, thus further promoting reproductive fitness even at high age.

Over the past decade, evidence has accumulated that new protein-coding genes can emerge de novo from previously non-coding DNA. Most studies have focused on large scale computational predictions of de novo protein-coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins are scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are short and predicted to be disordered. Here, we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli. We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains, and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30% to 62%. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST-tag with T7 Express cells and co-expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express. STATEMENT: Today, we know that proteins do not only evolve by duplication and divergence of existing proteins but also arise from previously non-coding DNA. These proteins are called de novo proteins. Their properties are still poorly understood and their experimental analysis faces major obstacles. Here, we aim to present a starting point for soluble expression of de novo proteins with the help of chaperones and thereby enable further characterization.

Global climate change can influence organismic interactions like those between hosts and parasites. Rising temperatures may exacerbate the exploitation of hosts by parasites, especially in ectothermic systems. The metabolic activity of ectotherms is strongly linked to temperature and generally increases when temperatures rise. We hypothesized that temperature change in combination with parasite infection interferes with the host's immunometabolism. We used a parasite, the avian cestode Schistocephalus solidus, which taps most of its resources from the metabolism of an ectothermic intermediate host, the three-spined stickleback. We experimentally exposed sticklebacks to this parasite, and studied liver transcriptomes 50 days after infection at 13°C and 24°C, to assess their immunometabolic responses. Furthermore, we monitored fitness param-eters of the parasite and examined immunity and body condition of the sticklebacks at 13°C, 18°C and 24°C after 36, 50 and 64 days of infection. At low temperatures (13°C), S. solidus growth was constrained, presumably also by the more active stickle-back's immune system, thus delaying its infectivity for the final host to 64 days. Warmer temperature (18°C and 24°C) enhanced S. solidus growth, and it became infective to the final host already after 36 days. Overall, S. solidus produced many more viable off-spring after development at elevated temperatures. In contrast, stickleback hosts had lower body conditions, and their immune system was less active at warm temperature. The stickleback's liver transcriptome revealed that mainly metabolic processes were differentially regulated between temperatures, whereas immune genes were not strongly affected. Temperature effects on gene expression were strongly enhanced in infected sticklebacks, and even in exposed-but-not-infected hosts. These data suggest that the parasite exposure in concert with rising temperature, as to be expected with global climate change, shifted the host's immunometabolism, thus providing nutrients for the enormous growth of the parasite and, at the same time suppressing immune defence.

In addition to known genes, much of the human genome is transcribed into RNA. Chance formation of novel open reading frames (ORFs) can lead to the translation of myriad new proteins. Some of these ORFs may yield advantageous adaptive de novo proteins. However, widespread translation of noncoding DNA can also produce hazardous protein molecules, which can misfold and/or form toxic aggregates. The dynamics of how de novo proteins emerge from potentially toxic raw materials and what influences their long-term survival are unknown. Here, using transcriptomic data from human and five other primates, we generate a set of transcribed human ORFs at six conservation levels to investigate which properties influence the early emergence and long-term retention of these expressed ORFs. As these taxa diverged from each other relatively recently, we present a fine scale view of the evolution of novel sequences over recent evolutionary time. We find that novel human-restricted ORFs are preferentially located on GC-rich gene-dense chromosomes, suggesting their retention is linked to pre-existing genes. Sequence properties such as intrinsic structural disorder and aggregation propensity—which have been proposed to play a role in survival of de novo genes—remain unchanged over time. Even very young sequences code for proteins with low aggregation propensities, suggesting that genomic regions with many novel transcribed ORFs are concomitantly less likely to produce ORFs which code for harmful toxic proteins. Our data indicate that the survival of these novel ORFs is largely stochastic rather than shaped by selection.

Eukaryotic genomes frequently acquire new protein-coding genes which may significantly impact an organism’s fitness. Novel genes can be created, for example, by duplication of large genomic regions or de novo, from previously non-coding DNA. Either way, creation of a novel transcript is an essential early step during novel gene emergence. Most studies on the gain-and-loss dynamics of novel genes so far have compared genomes between species, constraining analyses to genes that have remained fixed over long time scales. However, the importance of novel genes for rapid adaptation among populations has recently been shown. Therefore, since little is known about the evolutionary dynamics of transcripts across natural populations, we here study transcriptomes from several tissues and nine geographically distinct populations of an ecological model species, the three-spined stickleback. Our findings suggest that novel genes typically start out as transcripts with low expression and high tissue specificity. Early expression regulation appears to be mediated by gene-body methylation. Although most new and narrowly expressed genes are rapidly lost, those that survive and subsequently spread through populations tend to gain broader and higher expression levels. The properties of the encoded proteins, such as disorder and aggregation propensity, hardly change. Correspondingly, young novel genes are not preferentially under positive selection but older novel genes more often overlap with FST outlier regions. Taken together, expression of the surviving novel genes is rapidly regulated, probably via epigenetic mechanisms, while structural properties of encoded proteins are non-debilitating and might only change much later.

Orphan genes, lacking detectable homologs in outgroup species, typically represent 10–30% of eukaryotic genomes. Efforts to find the source of these young genes indicate that de novo emergence from non-coding DNA may in part explain their prevalence. Here, we investigate the roots of orphan gene emergence in the Drosophila genus. Across the annotated proteomes of twelve species, we find 6297 orphan genes within 4953 taxon-specific clusters of orthologs. By inferring the ancestral DNA as non-coding for between 550 and 2467 (8.7–39.2%) of these genes, we describe for the first time how de novo emergence contributes to the abundance of clade-specific Drosophila genes. In support of them having functional roles, we show that de novo genes have robust expression and translational support. However, the distinct nucleotide sequences of de novo genes, which have characteristics intermediate between intergenic regions and conserved genes, reflect their recent birth from non-coding DNA. We find that de novo genes encode more disordered proteins than both older genes and intergenic regions. Together, our results suggest that gene emergence from non-coding DNA provides an abundant source of material for the evolution of new proteins. Following gene birth, gradual evolution over large evolutionary timescales moulds sequence properties towards those of conserved genes, resulting in a continuum of properties whose starting points depend on the nucleotide sequences of an initial pool of novel genes. Electronic supplementary material The online version of this article (10.1007/s00239-020-09939-z) contains supplementary material, which is available to authorized users.

Balancing selection describes evolutionary processes that maintain genetic diversity. To date, the number of impacted genes and underlying biological functions remain elusive. Using 60 three-spined stickleback genomes (Gasterosteus aculeatus) from five recently diverged lake-river population-pairs, we performed genome-wide scans across two levels of organization: population-pairs and populations. We overlapped Tajima’s D and Watterson’s estimator metrics and verified signals with additional summary statistics, and evaluated alternative explanations: neutral evolution, population structure, associative overdominance, or demographic change. Candidate windows exhibiting signals of balancing selection spanned 2.31% (population-pair) and 3.10% (population) of the autosomes. These candidate windows had extended linkage disequilibrium and were enriched in intergenic and non-synonymous SNPs. We identified 715 (population-pair) and 1,010 (population) candidate genes under balancing selection. Importantly, using conservative thresholds, we found a small proportion of candidate genes overlapped with highly differentiated loci or regions of potential associative overdominance. There was little evidence of confounding effects originating from demographic change. Overall, candidate genes under balancing selection were associated with functions related to interactions with the environment (olfaction and receptor signalling pathways). Our results demonstrate selection that maintains standing genetic variation is common and evolves in response to local environmental pressures, playing an important role in adaptation.

Over the past decade, evidence has accumulated that new protein coding genes can emerge de novo from previously non-coding DNA. Most studies have focused on large scale computational predictions of de novo protein coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins is scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are predicted to be short and disordered. Here we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli.We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30 % to 62 %. Our findings indicate that the best combination for expressing putative de novo proteins in E.coli is a GST-tag with T7 Express cells and co-expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.

Insects provide an unparalleled opportunity to link genomic changes with the rise of novel phenotypes, given tremendous variation in the numerous and complex adaptations displayed across the group. Among these numerous and complex adaptations, live-birth has arisen repeatedly and independently in insects and across the tree of life, suggesting this is one of the most common types of convergent evolution among animals. We sequenced the genome and transcriptome of the Pacific beetle-mimic cockroach, the only truly viviparous cockroach, and performed comparative analyses including two other viviparous insect lineages, the tsetse and aphids, to unravel the genomic basis underlying the transition to viviparity in insects. We identified pathways experiencing adaptive evolution, common in all viviparous insects surveyed, involved in uro-genital remodeling, maternal control of embryo development, tracheal system, and heart development. Our findings suggest the essential role of those pathways for the development of placenta-like structure enabling embryo development and nutrition. Viviparous transition seems also to be accompanied by the duplication of genes involved in eggshell formation. Our findings from the viviparous cockroach and tsetse reveal that genes involved in uterine remodeling are up-regulated and immune genes are down-regulated during the course of pregnancy. These changes may facilitate structural changes to accommodate developing young and protect them from the mothers immune system. Our results denote a convergent evolution of live-bearing in insects and suggest similar adaptive mechanisms occurred in vertebrates, targeting pathways involved in eggshell formation, uro-genital remodeling, enhanced tracheal and heart development, and reduced immunity.

Final Supplemental Data "New genomic signals underlying the emergence of human proto-genes"

De novo genes are novel genes which emerge from non-coding DNA. Until now, little is known about de novo genes properties, correlated to their age and mechanisms of emergence. In this study, we investigate four properties: introns, upstream regulatory motifs, 5’ UTRs and protein domains, in 23135 human proto-genes. We found that proto-genes contain introns, whose number and position correlates with the genomic position of proto-gene emergence. The origin of these introns is debated, as our result suggest that 41% proto-genes might have captured existing introns, as well as the fact that 13.7% of them do not splice the ORF. We show that proto-genes which emerged via overprinting tend to be more enriched in core promotor motifs, while intergenic and intronic ones are more enriched in enhancers, even if the motif TATA is most expressed upstream these genes. Intergenic and intronic 5’ UTRs of protogenes have a lower potential to stabilise mRNA structures than exonic proto-genes and established human genes. Finally, we confirm that proto-genes gain new putative domains with age. Overall, we find that regulatory motifs inducing transcription and translation of previously non-coding sequences may facilitate proto-gene emergence. Our paper demonstrates that introns, 5’UTRs, and domains have specific properties in proto-genes. We also show the importance of studying proto-genes in relation to their genomic position, as it strongly impacts these properties.

De novo genes are novel genes which emerge from non-coding DNA. Until now, little is known about de novo genes properties, correlated to their age and mechanisms of emergence. In this study, we investigate four properties: introns, upstream regulatory motifs, 5’ UTRs and protein domains in 23135 human proto-genes. We found that proto-genes contain introns, whose number and position correlates with the genomic position of proto-gene emergence. The origin of these introns is debated, as our result suggest that 41% proto-genes might have captured existing introns, as well as the fact that 13.7% of them do not splice the ORF. We show that proto-genes which emerged via overprinting tend to be more enriched in core promotor motifs, even if the motif TATA is most expressed upstream these genes, while intergenic and intronic ones are more enriched in enhancers. Intergenic and intronic 5’ UTRs of proto-genes have a lower potential to stabilise mRNA structures than exonic proto-genes and established human genes. Finally, we confirm that proto-genes gain new putative domains with age. Overall, we find that regulatory motifs inducing transcription and translation of previously non-coding sequences may facilitate proto-gene emergence. Our paper demonstrates that introns, 5’UTRs, and domains have specific properties in proto-genes. We also show the importance of studying proto-genes in relation to their genomic position, as it strongly impacts these properties.

Balancing selection describes evolutionary processes that maintain genetic diversity. To date, the number of impacted genes and underlying biological functions remain elusive. Using 60 three-spined stickleback genomes (Gasterosteus aculeatus) from five recently diverged lake-river population-pairs, we performed genome-wide scans across two levels of organization: population-pairs and populations. We overlapped Tajima’s D and Watterson’s estimator metrics and verified signals with additional summary statistics, and evaluated alternative explanations: neutral evolution, population structure, associated overdominance, or demographic change. Candidate windows exhibiting signals of balancing selection spanned 2.31% (population-pair) and 3.10% (population) of the autosomes. These candidate windows had extended linkage disequilibrium and were enriched in intergenic and non-synonymous SNPs. We identified 715 (population-pair) and 1,010 (population) candidate genes under balancing selection. Importantly, using conservative thresholds, we found a small proportion of candidate genes overlapped with highly differentiated loci or regions of potential associated overdominance. There was little evidence of confounding effects originating from demographic change. Overall, candidate genes under balancing selection were associated with functions related to interactions with the environment (olfaction and receptor signalling pathways). Our results demonstrate selection that maintains standing genetic variation is common and evolves in response to local environmental pressures, playing an important role in adaptation.

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it., Séité, Harrison, et al. investigate the mechanisms underlying long lifespan in queens and kings of the highly social termite Macrotermes natalensis. Using transcriptomics, lipidomics and metabolomics, this study shows that several aging-related processes are reduced in the fat bodies of these reproductives and that an upregulated insulin-like peptide, Ilp9, does not lead to deleterious fat storage in old queens, while simple sugars dominate in their hemolymph.

Evolutionary relationships of protein families can be characterized either by networks or by trees. Whereas trees allow for hierarchical grouping and reconstruction of the most likely ancestral sequences, networks lack a time axis but allow for thresholds of pairwise sequence identity to be chosen and, therefore, the clustering of family members with presumably more similar functions. Here, we use the large family of arylsulfatases and phosphonate monoester hydrolases to investigate similarities, strengths and weaknesses in tree and network representations. For varying thresholds of pairwise sequence identity, values of betweenness centrality and clustering coefficients were derived for nodes of the reconstructed ancestors to measure the propensity to act as a bridge in a network. Based on these properties, ancestral protein sequences emerge as bridges in protein sequence networks. Interestingly, many ancestral protein sequences appear close to extant sequences. Therefore, reconstructed ancestor sequences might also be interpreted as yet-to-be-identified homologues. The concept of ancestor reconstruction is compared to consensus sequences, too. It was found that hub sequences in a network, e.g. reconstructed ancestral sequences that are connected to many neighbouring sequences, share closer similarity with derived consensus sequences. Therefore, some reconstructed ancestor sequences can also be interpreted as consensus sequences.

The ecological success of social Hymenoptera (ants, bees, wasps) depends on the division of labour between the queen and workers. Each caste is highly specialised in its respective function in morphology, behaviour and life-history traits, such as lifespan and fecundity. Despite strong defences against alien intruders, insect societies are vulnerable to social parasites, such as workerless inquilines or slave-making (dulotic) ants. Here, we investigate whether gene expression varies in parallel ways between lifestyles (slave-making versus host ants) across five independent origins of ant slavery in the “Formicoxenus-group” of the ant tribe Crematogastrini. As caste differences are often less pronounced in slave-making ants than non-parasitic ants, we also compare the transcriptomes of queens and workers in these species. We demonstrate a substantial overlap in expression differences between queens and workers across taxa, irrespective of lifestyle. Caste affects the transcriptomes much more profoundly than lifestyle, as indicated by 37 times more genes being linked to caste than to lifestyle and by multiple caste-associated gene modules with strong connectivity. However, several genes and one gene module are linked to the slave-making lifestyle across the independent origins, pointing to some evolutionary convergence. Finally, we do not find evidence for an interaction between caste and lifestyle, indicating that caste differences remain consistent even when species switch to a parasitic lifestyle. Our findings are a strong indication for the existence of a core set of genes whose expression is linked to the queen and worker caste in this ant taxon, supporting the “genetic toolkit” hypothesis.

lncRNAs are at the core of many regulatory processes and have also been recognized to be involved in various complex diseases. They affect gene regulation through direct interactions with RNA, DNA or proteins. Accordingly, lncRNAs structure is likely to be essential for their regulatory function. Point mutations, which manifest as SNPs (single nucleotide polymorphisms) in genome screens, can substantially alter their function and, subsequently, the expression of their down-stream regulated genes. To test the effect of SNPs on structure, we investigated lncRNAs associated with dilated cardiomyopathy. Among 322 human candidate lncRNAs we demonstrate first the significant association of a SNP located in lncRNA H19 using data from 1084 diseased and 751 control patients. H19 is generally highly expressed in the heart, with a complex expression pattern during heart development. Next, we used MFE (minimum free energy) folding to demonstrate a significant refolding in the secondary structure of this 861 nt long lncRNA. Since MFE folding may overlook the importance of sub-optimal structures, we showed that this refolding also manifests in the overall Boltzmann structure ensemble. There, the composition of structures is tremendously affected in their thermodynamic probabilities through the genetic variant. Finally, we confirmed these results experimentally, using SHAPE-Seq, corroborating that SNPs affecting such structures may explain hidden genetic variance not accounted for through genome wide association studies. Our results suggest that structural changes in lncRNAs, and lncRNA H19 in particular, affect regulatory processes and represent optimal targets for further in-depth studies probing their molecular interactions.

Even in the era of next generation sequencing, in which bioinformatics tools abound, annotating transcriptomes and proteomes remains a challenge. This can have major implications for the reliability of studies based on these datasets. Therefore, quality assessment represents a crucial step prior to downstream analyses on novel transcriptomes and proteomes. DOGMA allows such a quality assessment to be carried out. The data of interest are evaluated based on a comparison with a core set of conserved protein domains and domain arrangements. Depending on the studied species, DOGMA offers precomputed core sets for different phylogenetic clades. We now developed a web server for the DOGMA software, offering a user-friendly, simple to use interface. Additionally, the server provides a graphical representation of the analysis results and their placement in comparison to publicly available data. The server is freely available under https://domainworld-services.uni-muenster.de/dogma/. Additionally, for large scale analyses the software can be downloaded free of charge from https://domainworld.uni-muenster.de.

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ∼15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.Author SummaryGenomes are in flux, as genes are constantly added and lost throughout evolution. New genes were once thought to arise almost exclusively via the modification or duplication of existing genes. Recently, however, interest has grown in alternative modes of new gene origination, such as de novo evolution from genetic material that previously did not encode proteins. Many de novo genes are expressed in male reproductive tissues, but their significance for fertility is not well understood. We screened likely de novo genes expressed in the Drosophila testis for reproductive roles and found one gene, atlas, essential for male fertility. We leveraged genetic and cell biological experiments to investigate roles for Atlas protein in reproduction and found that it is required during sperm development for proper packaging of DNA in the sperm nucleus. Evolutionary analyses of this gene revealed a complicated history, including loss in some lineages, movement between chromosomes, and fusion with a non-protein-coding gene. Studying both the functions and evolutionary histories of new proteins illustrates how they might evolve critical roles in biological processes despite their relative novelty. Furthermore, the study of atlas identifies an essential genetic player in the fly testis, an important model system for understanding how gametes are produced.

Evolutionary theories of ageing predict a reduction in selection efficiency with age, a so-called ‘selection shadow’, due to extrinsic mortality decreasing effective population size with age. Classic symptoms of ageing include a deterioration in transcriptional regulation and protein homeostasis. Understanding how ant queens defy the trade-off between fecundity and lifespan remains a major challenge for the evolutionary theory of ageing. It has often been discussed that the low extrinsic mortality of ant queens, that are generally well protected within the nest by workers and soldiers, should reduce the selection shadow acting on old queens. We tested this by comparing strength of selection acting on genes upregulated in young and old queens of the ant, Cardiocondyla obscurior. In support of a reduced selection shadow, we find old-biased genes to be under strong purifying selection. We also analysed a gene co-expression network (GCN) with the aim to detect signs of ageing in the form of deteriorating regulation and proteostasis. We find no evidence for ageing. In fact, we detect higher connectivity in old queens indicating increased transcriptional regulation with age. Within the GCN, we discover five highly correlated modules that are upregulated with age. These old-biased modules regulate several anti-ageing mechanisms such as maintenance of proteostasis, transcriptional regulation and stress response. We observe stronger purifying selection on central hub genes of these old-biased modules compared to young-biased modules. These results indicate a lack of transcriptional ageing in old C. obscurior queens possibly facilitated by strong selection at old age and well-regulated anti-ageing mechanisms.Significance StatementUnderstanding the exceptional longevity of ant queens and how they defy the trade-off between fecundity and lifespan remains a major challenge for the evolutionary theory and molecular biology of ageing. In this study we offer several clues as to how this occurs on a molecular level in C. obscurior queens. Specifically, we believe a reduction in the selection shadow due to low extrinsic mortality, has allowed the evolution of well-regulated anti-ageing mechanisms. Consequently, we suggest several promising starting points for future research into the poorly understood phenomenon of extreme longevity in ant queens. Making progress in this field will not only allow us to better understand longevity and fertility in social insects but may also offer interesting research strategies for human ageing.

Comparative genomic studies have repeatedly shown that new protein-coding genes can emerge de novo from non-coding DNA. Still unknown is how and when the structures of encoded de novo proteins emerge and evolve. Combining biochemical, genetic and evolutionary analyses, we elucidate the function and structure of goddard, a gene which appears to have evolved de novo at least 50 million years ago within the Drosophila genus.Previous studies found that goddard is required for male fertility. Here, we show that Goddard protein localizes to elongating sperm axonemes and that in its absence, elongated spermatids fail to undergo individualization. Combining modelling, NMR and CD data, we show that Goddard protein contains a large central α-helix, but is otherwise partially disordered. We find similar results for Goddard’s orthologs from divergent fly species and their reconstructed ancestral sequences. Accordingly, Goddard’s structure appears to have been maintained with only minor changes over millions of years.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.