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Speciation is responsible for the vast diversity of life, and hybrid inviability, by reducing gene flow between populations, is a major contributor to this process. In the parasitoid wasp genus Nasonia, F2 hybrid males of Nasonia vitripennis and Nasonia giraulti experience an increased larval mortality rate relative to the parental species. Previous studies indicated that this increase of mortality is a consequence of incompatibilities between multiple nuclear loci and cytoplasmic factors of the parental species, but could only explain ∼40% of the mortality rate in hybrids with N. giraulti cytoplasm. Here we report a locus on chromosome 5 that can explain the remaining mortality in this cross. We show that hybrid larvae that carry the incompatible allele on chromosome 5 halt growth early in their development and that ∼98% die before they reach adulthood. On the basis of these new findings, we identified a nuclear-encoded OXPHOS gene as a strong candidate for being causally involved in the observed hybrid breakdown, suggesting that the incompatible mitochondrial locus is one of the six mitochondrial-encoded NADH genes. By identifying both genetic and physiological mechanisms that reduce gene flow between species, our results provide valuable and novel insights into the evolutionary dynamics of speciation. [ABSTRACT FROM AUTHOR]

The occurrence of hybrid incompatibilities forms an important stage during the evolution of reproductive isolation. In early stages of speciation, males and females often respond differently to hybridization. Haldane's rule states that the heterogametic sex suffers more from hybridization than the homogametic sex. Although haplodiploid reproduction (haploid males, diploid females) does not involve sex chromosomes, sex-specific incompatibilities are predicted to be prevalent in haplodiploid species. Here, we evaluate the effect of sex/ploidy level on hybrid incompatibilities and locate genomic regions that cause increased mortality rates in hybrid males of the haplodiploid wasps Nasonia vitripennis and Nasonia longicornis. Our data show that diploid F1 hybrid females suffer less from hybridization than haploid F2 hybrid males. The latter not only suffer from an increased mortality rate, but also from behavioural and spermatogenic sterility. Genetic mapping in recombinant F2 male hybrids revealed that the observed hybrid mortality is most likely due to a disruption of cytonuclear interactions. As these sex-specific hybrid incompatibilities follow predictions based on Haldane's rule, our data accentuate the need to broaden the view of Haldane's rule to include species with haplodiploid sex determination, consistent with Haldane's original definition. [ABSTRACT FROM AUTHOR]

Many insects rely on cuticular hydrocarbons (CHCs) as major recognition signals between individuals. Previous research on the genetics of CHCs has focused on Drosophila in which the roles of three desaturases and one elongase were highlighted. Comparable studies in other insect taxa have not been conducted so far. Here, we explore the genetics of CHCs in hybrids of the jewel wasps Nasonia giraulti and Nasonia vitripennis. We analyzed the CHC profiles of pure strain and of F2 hybrid males using gas chromatography coupled with mass spectrometry and distinguished 54 peaks, of which we identified 52 as straight-chain, monounsaturated, or methyl-branched CHCs. The latter compound class proved to be particularly abundant and diverse in Nasonia. Quantitative trait locus (QTL) analysis suggests fixed genetic differences between the two strains in 42 of the 54 studied traits, making Nasonia a promising genetic model for identifying genes involved in CHC biosynthesis. QTL for methyl-branched CHCs partly clustered in genomic regions with high recombination rate: a possible indication for pleiotropic genes that control their biosynthesis, which is largely unexplored so far. Finally, we identified and mapped genes in the Nasonia genome with high similarity to genes that have been implicated in alkene biosynthesis in Drosophila and discuss those that match in their position with predicted QTL for alkenes. [ABSTRACT FROM AUTHOR]

Here we present a nearly complete species-level phylogeny including 23 of the 25 known species of the forest-dwelling herbivorous scarab chafer beetle genus Pleophylla (Coleoptera: Scarabaeidae: Sericinae), based on the analysis of 950 nuclear genes (metazoan-level universal single-copy orthologs; mzl-USCOs). DNA sequences were obtained from freshly collected, ethanol-preserved samples and from dried museum specimens by target enrichment or genome shotgun sequencing. Alignment completeness of mzl-USCOs newly obtained here by target DNA enrichment of ethanol samples were very heterogenous and lower (29-62 %) than in Dietz et al. (2023a), while that of sequences recovered from dried samples was even lower (∼19 %). Alignment completeness of the sequences obtained from low coverage shotgun sequencing was highest (∼92 %), although the average coverage was much lower than for the target enrichment samples. We used the resulting phylogeny to reconstruct the historical biogeography of the group. To estimate a time-calibrated tree, we combined the mzl-USCO data of Pleophylla with a nucleotide alignment from an available transcriptomic dataset of Scarabaeoidea and used two different sets of secondary calibration points. Despite the problems associated with the capture rate of mzl-USCO sequences from museum specimens, we were able to infer a well-resolved phylogeny of the genus Pleophylla that also provided reliable estimates of the phylogenetic position of species for which we had little sequence data. Our study clearly identified South Africa as the geographic origin of Pleophylla. Timing and biogeographic history confirm a persistent fragmentation of forests since the Eocene. The occurrence of only one long-distance dispersal event from southern Africa to the Eastern African Arc even during the Miocene highlights the limited dispersal possibilities for these forest-adapted chafers, which do not seem to have had important northerly range expansions along hypothetical forest corridors during the Pleistocene., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Caddisflies (Trichoptera) are among the most diverse groups of freshwater animals with more than 16 000 described species. They play a fundamental role in freshwater ecology and environmental engineering in streams, rivers and lakes. Because of this, they are frequently used as indicator organisms in biomonitoring programmes. Despite their importance, key questions concerning the evolutionary history of caddisflies, such as the timing and origin of larval case making, remain unanswered owing to the lack of a well-resolved phylogeny. Here, we estimated a phylogenetic tree using a combination of transcriptomes and targeted enrichment data for 207 species, representing 48 of 52 extant families and 174 genera. We calibrated and dated the tree with 33 carefully selected fossils. The first caddisflies originated approximately 295 million years ago in the Permian, and major suborders began to diversify in the Triassic. Furthermore, we show that portable case making evolved in three separate lineages, and shifts in diversification occurred in concert with key evolutionary innovations beyond case making.

Polyandry, the practice of females mating with multiple males, is a strategy found in many insect groups. Whether it increases the likelihood of receiving beneficial genes from male partners and other potential benefits for females is controversial. Strepsiptera are generally considered monandrous, but in a few species females have been observed copulating serially with multiple males. Here we show that the offspring of a single female can have multiple fathers in two Strepsiptera species: Stylops ovinae (Stylopidae) and Xenos vesparum (Xenidae). We studied female polyandry in natural populations of these two species by analysis of polymorphic microsatellite loci. Our results showed that several fathers can be involved in both species, in some cases up to four. Mating experiments with S. ovinae have shown that the first male to mates with a given female contributes to a higher percentage of the offspring than subsequent males. In X. vesparum, however, we found no significant correlation between mating duration and offspring contribution. The prolonged copulation observed in S. ovinae may have the advantage of reducing competition with sperm from other males. Our results show that monandry may not be the general pattern of reproduction in the insect order Strepsiptera., (© 2024. The Author(s).)

Metazoa-level universal single-copy orthologs (mzl-USCOs) are universally applicable markers for DNA taxonomy in animals that can replace or supplement single-gene barcodes. Previously, mzl-USCOs from target enrichment data were shown to reliably distinguish species. Here, we tested whether USCOs are an evenly distributed, representative sample of a given metazoan genome and therefore able to cope with past hybridization events and incomplete lineage sorting. This is relevant for coalescent-based species delimitation approaches, which critically depend on the assumption that the investigated loci do not exhibit autocorrelation due to physical linkage. Based on 239 chromosome-level assembled genomes, we confirmed that mzl-USCOs are genetically unlinked for practical purposes and a representative sample of a genome in terms of reciprocal distances between USCOs on a chromosome and of distribution across chromosomes. We tested the suitability of mzl-USCOs extracted from genomes for species delimitation and phylogeny in four case studies: Anopheles mosquitos, Drosophila fruit flies, Heliconius butterflies and Darwin's finches. In almost all instances, USCOs allowed delineating species and yielded phylogenies that corresponded to those generated from whole genome data. Our phylogenetic analyses demonstrate that USCOs may complement single-gene DNA barcodes and provide more accurate taxonomic inferences. Combining USCOs from sources that used different versions of ortholog reference libraries to infer marker orthology may be challenging and, at times, impact taxonomic conclusions. However, we expect this problem to become less severe as the rapidly growing number of reference genomes provides a better representation of the number and diversity of organismal lineages., (© 2023 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Cuticular hydrocarbons (CHCs) cover the cuticle of insects and serve as desiccation barrier and as semiochemicals. While the main enzymatic steps of CHC biosynthesis are well understood, few of the underlying genes have been identified. Here we show how exploitation of intrasexual CHC dimorphism in a mason wasp, Odynerus spinipes, in combination with whole-genome sequencing and comparative transcriptomics facilitated identification of such genes. RNAi-mediated knockdown of twelve candidate gene orthologs in the honey bee, Apis mellifera, confirmed nine genes impacting CHC profile composition. Most of them have predicted functions consistent with current knowledge of CHC metabolism. However, we found first-time evidence for a fatty acid amide hydrolase also influencing CHC profile composition. In situ hybridization experiments furthermore suggest trophocytes participating in CHC biosynthesis. Our results set the base for experimental CHC profile manipulation in Hymenoptera and imply that the evolutionary origin of CHC biosynthesis predates the arthropods' colonization of land., (© 2023. The Author(s).)

The extent of interspecific gene flow and its consequences for the initiation, maintenance, and breakdown of species barriers in natural systems remain poorly understood. Interspecific gene flow by hybridization may weaken adaptive divergence, but can be overcome by selection against hybrids, which may ultimately promote reinforcement. An informative step towards understanding the role of gene flow during speciation is to describe patterns of past gene flow among extant species. We investigate signals of admixture between allopatric and sympatric populations of the two closely related European dung fly species Sepsis cynipsea and S. neocynipsea (Diptera: Sepsidae). Based on microsatellite genotypes, we first inferred a baseline demographic history using Approximate Bayesian Computation. We then used genomic data from pooled DNA of natural and laboratory populations to test for past interspecific gene flow based on allelic configurations discordant with the inferred population tree (ABBA-BABA test with D -statistic). Comparing the detected signals of gene flow with the contemporary geographic relationship among interspecific pairs of populations (sympatric vs. allopatric), we made two contrasting observations. At one site in the French Cevennes, we detected an excess of past interspecific gene flow, while at two sites in Switzerland we observed lower signals of past microsatellite genotypes gene flow among populations in sympatry compared to allopatric populations. These results suggest that the species boundaries between these two species depend on the past and/or present eco-geographic context in Europe, which indicates that there is no uniform link between contemporary geographic proximity and past interspecific gene flow in natural populations., Supplementary Information: The online version contains supplementary material available at 10.1007/s11692-023-09612-5., Competing Interests: Competing interestThe authors declare no competing interests., (© The Author(s) 2023.)

Background: Brood parasites can exert strong selection pressure on their hosts. Many brood parasites escape their detection by mimicking sensory cues of their hosts. However, there is little evidence whether or not the hosts are able to escape the parasites' mimicry by changing these cues. We addressed this question by analyzing cuticular hydrocarbon (CHC) profiles of Cerceris and Philanthus wasps and their brood parasites, cuckoo wasps mimicking the CHC profiles of their hosts. Some of these hosts use hydrocarbons to preserve their prey against fungal infestation and thus, they cannot significantly change their CHC composition in response to chemical mimicry by Hedychrum brood parasites., Results: We found that the CHC overlap between brood parasites and their hosts was lower in case of host wasps not preserving their prey than in case of prey-preserving host wasps, whose CHC evolution is constrained. Furthermore, the CHC profiles in non-preserving host wasps is more strongly diversified in females than in males, thus in the sex that is chemically mimicked by brood parasites., Conclusion: Our results provide evidence for a chemical arms race between those hosts that are liberated from stabilizing selection on their chemical template and their parasites., (© 2022. The Author(s).)

Traumatic insemination describes an unusual form of mating during which a male penetrates the body wall of its female partner to inject sperm. Females unable to prevent traumatic insemination have been predicted to develop either traits of tolerance or of resistance, both reducing the fitness costs associated with the male-inflicted injury. The evolution of tolerance traits has previously been suggested for the bed bug. Here we present data suggesting that tolerance traits also evolved in females of the twisted-wing parasite species Stylops ovinae and Xenos vesparum . Using micro-indentation experiments and confocal laser scanning microscopy, we found that females of both investigated species possess a uniform resilin-rich integument that is notably thicker at penetration sites than at control sites. As the thickened cuticle does not seem to hamper penetration by males, we hypothesise that thickening of the cuticle resulted in reduced penetration damage and loss of haemolymph and in improved wound sealing. To evaluate the evolutionary relevance of the Stylops -specific paragenital organ and penis shape variation in the context of inter- and intraspecific competition, we conducted attraction and interspecific mating experiments, as well as a geometric-morphometric analysis of S. ovinae and X. vesparum penises. We found that S. ovinae females indeed attract sympatrically distributed congeneric males. However, only conspecific males were able to mate. In contrast, we did not observe any heterospecific male attraction by Xenos females. We therefore hypothesise that the paragenital organ in the genus Stylops represents a prezygotic mating barrier that prevents heterospecific matings., Competing Interests: Stanislav Gorb is an Academic Editor for PeerJ., (© 2022 Jandausch et al.)

Cuticular hydrocarbons (CHCs) serve two fundamental functions in insects: protection against desiccation and chemical signalling. How the interaction of genes shapes CHC profiles, which are essential for insect survival, adaptation and reproductive success, is still poorly understood. Here we investigate the genetic and genomic basis of CHC biosynthesis and variation in parasitoid wasps of the genus Nasonia . We mapped 91 quantitative trait loci (QTL) explaining the variation of a total of 43 CHCs in F 2 hybrid males from interspecific crosses between three Nasonia species. To identify candidate genes, we localized orthologues of CHC biosynthesis-related genes in the Nasonia genomes. We discovered multiple genomic regions where the location of QTL coincides with the location of CHC biosynthesis-related candidate genes. Most conspicuously, on a region close to the centromere of chromosome 1, multiple CHC biosynthesis-related candidate genes co-localize with several QTL explaining variation in methyl-branched alkanes. The genetic underpinnings behind this compound class are not well understood so far, despite their high potential for encoding chemical information as well as their prevalence in hymenopteran CHC profiles. Our study considerably extends our knowledge on the genetic architecture governing this important compound class, establishing a model for methyl-branched alkane genetics in the Hymenoptera in general.

Dragonflies and damselflies are among the earliest flying insects with extant representatives. However, unraveling details of their long evolutionary history, such as egg laying (oviposition) strategies, is impeded by unresolved phylogenetic relationships, particularly in damselflies. Here we present a transcriptome-based phylogenetic reconstruction of Odonata, analyzing 2,980 protein-coding genes in 105 species representing nearly all the order's families. All damselfly and most dragonfly families are recovered as monophyletic. Our data suggest a sister relationship between dragonfly families of Gomphidae and Petaluridae. According to our divergence time estimates, both crown-Zygoptera and -Anisoptera arose during the late Triassic. Egg-laying with a reduced ovipositor apparently evolved in dragonflies during the late Jurassic/early Cretaceous. Lastly, we also test the impact of fossil choice and placement, particularly, of the extinct fossil species, † Triassolestodes asiaticus , and † Proterogomphus renateae on divergence time estimates. We find placement of † Proterogomphus renateae to be much more impactful than † Triassolestodes asiaticus ., Competing Interests: Authors declare no competing interests., (© 2021 The Authors.)

Chemoreceptors help insects to interact with their environment, to detect and assess food sources and oviposition sites, and to aid in intra- and interspecific communication. In Hymenoptera, species of eusocial lineages possess large chemoreceptor gene repertoires compared with solitary species, possibly because of their additional need to recognize nest-mates and caste. However, a critical piece of information missing so far has been the size of chemoreceptor gene repertoires of solitary apoid wasps. Apoid wasps are a paraphyletic group of almost exclusively solitary Hymenoptera phylogenetically positioned between ant and bee, both of which include eusocial species. We report the chemosensory-related gene repertoire sizes of three apoid wasps: Ampulex compressa, Cerceris arenaria, and Psenulus fuscipennis. We annotated genes encoding odorant (ORs), gustatory, and ionotropic receptors and chemosensory soluble proteins and odorant-binding proteins in transcriptomes of chemosensory tissues of the above three species and in early draft genomes of two species, A. compressa and C. arenaria. Our analyses revealed that apoid wasps possess larger OR repertoires than any bee lineage, that the last common ancestor of Apoidea possessed a considerably larger OR repertoire (∼160) than previously estimated (73), and that the expansion of OR genes in eusocial bees was less extensive than previously assumed. Intriguingly, the evolution of pollen-collecting behavior in the stem lineage of bees was associated with a notable loss of OR gene diversity. Thus, our results support the view that herbivorous Hymenoptera tend to possess smaller OR repertoires than carnivorous, parasitoid, or kleptoparasitic species., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: The most species-rich radiation of animal life in the 66 million years following the Cretaceous extinction event is that of schizophoran flies: a third of fly diversity including Drosophila fruit fly model organisms, house flies, forensic blow flies, agricultural pest flies, and many other well and poorly known true flies. Rapid diversification has hindered previous attempts to elucidate the phylogenetic relationships among major schizophoran clades. A robust phylogenetic hypothesis for the major lineages containing these 55,000 described species would be critical to understand the processes that contributed to the diversity of these flies. We use protein encoding sequence data from transcriptomes, including 3145 genes from 70 species, representing all superfamilies, to improve the resolution of this previously intractable phylogenetic challenge., Results: Our results support a paraphyletic acalyptrate grade including a monophyletic Calyptratae and the monophyly of half of the acalyptrate superfamilies. The primary branching framework of Schizophora is well supported for the first time, revealing the primarily parasitic Pipunculidae and Sciomyzoidea stat. rev. as successive sister groups to the remaining Schizophora. Ephydroidea, Drosophila's superfamily, is the sister group of Calyptratae. Sphaeroceroidea has modest support as the sister to all non-sciomyzoid Schizophora. We define two novel lineages corroborated by morphological traits, the 'Modified Oviscapt Clade' containing Tephritoidea, Nerioidea, and other families, and the 'Cleft Pedicel Clade' containing Calyptratae, Ephydroidea, and other families. Support values remain low among a challenging subset of lineages, including Diopsidae. The placement of these families remained uncertain in both concatenated maximum likelihood and multispecies coalescent approaches. Rogue taxon removal was effective in increasing support values compared with strategies that maximise gene coverage or minimise missing data., Conclusions: Dividing most acalyptrate fly groups into four major lineages is supported consistently across analyses. Understanding the fundamental branching patterns of schizophoran flies provides a foundation for future comparative research on the genetics, ecology, and biocontrol.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: The latest advancements in DNA sequencing technologies have facilitated the resolution of the phylogeny of insects, yet parts of the tree of Holometabola remain unresolved. The phylogeny of Neuropterida has been extensively studied, but no strong consensus exists concerning the phylogenetic relationships within the order Neuroptera. Here, we assembled a novel transcriptomic dataset to address previously unresolved issues in the phylogeny of Neuropterida and to infer divergence times within the group. We tested the robustness of our phylogenetic estimates by comparing summary coalescent and concatenation-based phylogenetic approaches and by employing different quartet-based measures of phylogenomic incongruence, combined with data permutations., Results: Our results suggest that the order Raphidioptera is sister to Neuroptera + Megaloptera. Coniopterygidae is inferred as sister to all remaining neuropteran families suggesting that larval cryptonephry could be a ground plan feature of Neuroptera. A clade that includes Nevrorthidae, Osmylidae, and Sisyridae (i.e. Osmyloidea) is inferred as sister to all other Neuroptera except Coniopterygidae, and Dilaridae is placed as sister to all remaining neuropteran families. Ithonidae is inferred as the sister group of monophyletic Myrmeleontiformia. The phylogenetic affinities of Chrysopidae and Hemerobiidae were dependent on the data type analyzed, and quartet-based analyses showed only weak support for the placement of Hemerobiidae as sister to Ithonidae + Myrmeleontiformia. Our molecular dating analyses suggest that most families of Neuropterida started to diversify in the Jurassic and our ancestral character state reconstructions suggest a primarily terrestrial environment of the larvae of Neuropterida and Neuroptera., Conclusion: Our extensive phylogenomic analyses consolidate several key aspects in the backbone phylogeny of Neuropterida, such as the basal placement of Coniopterygidae within Neuroptera and the monophyly of Osmyloidea. Furthermore, they provide new insights into the timing of diversification of Neuropterida. Despite the vast amount of analyzed molecular data, we found that certain nodes in the tree of Neuroptera are not robustly resolved. Therefore, we emphasize the importance of integrating the results of morphological analyses with those of sequence-based phylogenomics. We also suggest that comparative analyses of genomic meta-characters should be incorporated into future phylogenomic studies of Neuropterida.

The ease of sequencing DNA barcodes promoted a species identification system universally applicable across animal phyla. However, relying on a single mitochondrial DNA fragment has a number of drawbacks that can mislead species delimitation and identification. Implementation of multiple nuclear markers would mitigate the limits of the current barcoding system if these markers are universally applicable across species, carry sufficient information to discriminate between closely related species, and if sequencing and analyzing these markers can be automatized. As sequencing costs continue to fall, we believe that the time is right to extend DNA barcoding. Here we argue that nearly universal single-copy nuclear protein-coding genes deliver the desired characteristics and could be used to reliably delimit and identify animal species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods., Results: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception., Conclusions: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

The dipluran two-pronged bristletail Campodea augens is a blind ancestrally wingless hexapod with the remarkable capacity to regenerate lost body appendages such as its long antennae. As sister group to Insecta (sensu stricto), Diplura are key to understanding the early evolution of hexapods and the origin and evolution of insects. Here we report the 1.2-Gb draft genome of C. augens and results from comparative genomic analyses with other arthropods. In C. augens, we uncovered the largest chemosensory gene repertoire of ionotropic receptors in the animal kingdom, a massive expansion that might compensate for the loss of vision. We found a paucity of photoreceptor genes mirroring at the genomic level the secondary loss of an ancestral external photoreceptor organ. Expansions of detoxification and carbohydrate metabolism gene families might reflect adaptations for foraging behavior, and duplicated apoptotic genes might underlie its high regenerative potential. The C. augens genome represents one of the key references for studying the emergence of genomic innovations in insects, the most diverse animal group, and opens up novel opportunities to study the under-explored biology of diplurans., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles-remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: The location and modular structure of eukaryotic protein-coding genes in genomic sequences can be automatically predicted by gene annotation algorithms. These predictions are often used for comparative studies on gene structure, gene repertoires, and genome evolution. However, automatic annotation algorithms do not yet correctly identify all genes within a genome, and manual annotation is often necessary to obtain accurate gene models and gene sets. As manual annotation is time-consuming, only a fraction of the gene models in a genome is typically manually annotated, and this fraction often differs between species. To assess the impact of manual annotation efforts on genome-wide analyses of gene structural properties, we compared the structural properties of protein-coding genes in seven diverse insect species sequenced by the i5k initiative., Results: Our results show that the subset of genes chosen for manual annotation by a research community (3.5-7% of gene models) may have structural properties (e.g., lengths and exon counts) that are not necessarily representative for a species' gene set as a whole. Nonetheless, the structural properties of automatically generated gene models are only altered marginally (if at all) through manual annotation. Major correlative trends, for example a negative correlation between genome size and exonic proportion, can be inferred from either the automatically predicted or manually annotated gene models alike. Vice versa, some previously reported trends did not appear in either the automatic or manually annotated gene sets, pointing towards insect-specific gene structural peculiarities., Conclusions: In our analysis of gene structural properties, automatically predicted gene models proved to be sufficiently reliable to recover the same gene-repertoire-wide correlative trends that we found when focusing on manually annotated gene models only. We acknowledge that analyses on the individual gene level clearly benefit from manual curation. However, as genome sequencing and annotation projects often differ in the extent of their manual annotation and curation efforts, our results indicate that comparative studies analyzing gene structural properties in these genomes can nonetheless be justifiable and informative.

The beetle superfamily Dytiscoidea, placed within the suborder Adephaga, comprises six families. The phylogenetic relationships of these families, whose species are aquatic, remain highly contentious. In particular the monophyly of the geographically disjunct Aspidytidae (China and South Africa) remains unclear. Here we use a phylogenomic approach to demonstrate that Aspidytidae are indeed monophyletic, as we inferred this phylogenetic relationship from analyzing nucleotide sequence data filtered for compositional heterogeneity and from analyzing amino-acid sequence data. Our analyses suggest that Aspidytidae are the sister group of Amphizoidae, although the support for this relationship is not unequivocal. A sister group relationship of Hygrobiidae to a clade comprising Amphizoidae, Aspidytidae, and Dytiscidae is supported by analyses in which model assumptions are violated the least. In general, we find that both concatenation and the applied coalescent method are sensitive to the effect of among-species compositional heterogeneity. Four-cluster likelihood-mapping suggests that despite the substantial size of the dataset and the use of advanced analytical methods, statistical support is weak for the inferred phylogenetic placement of Hygrobiidae. These results indicate that other kinds of data (e.g. genomic meta-characters) are possibly required to resolve the above-specified persisting phylogenetic uncertainties. Our study illustrates various data-driven confounding effects in phylogenetic reconstructions and highlights the need for careful monitoring of model violations prior to phylogenomic analysis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Polyneoptera represents one of the major lineages of winged insects, comprising around 40,000 extant species in 10 traditional orders, including grasshoppers, roaches, and stoneflies. Many important aspects of polyneopteran evolution, such as their phylogenetic relationships, changes in their external appearance, their habitat preferences, and social behavior, are unresolved and are a major enigma in entomology. These ambiguities also have direct consequences for our understanding of the evolution of winged insects in general; for example, with respect to the ancestral habitats of adults and juveniles. We addressed these issues with a large-scale phylogenomic analysis and used the reconstructed phylogenetic relationships to trace the evolution of 112 characters associated with the external appearance and the lifestyle of winged insects. Our inferences suggest that the last common ancestors of Polyneoptera and of the winged insects were terrestrial throughout their lives, implying that wings did not evolve in an aquatic environment. The appearance of the first polyneopteran insect was mainly characterized by ancestral traits such as long segmented abdominal appendages and biting mouthparts held below the head capsule. This ancestor lived in association with the ground, which led to various specializations including hardened forewings and unique tarsal attachment structures. However, within Polyneoptera, several groups switched separately to a life on plants. In contrast to a previous hypothesis, we found that social behavior was not part of the polyneopteran ground plan. In other traits, such as the biting mouthparts, Polyneoptera shows a high degree of evolutionary conservatism unique among the major lineages of winged insects., Competing Interests: The authors declare no conflict of interest.

Background: Transposable elements (TEs) are a major component of metazoan genomes and are associated with a variety of mechanisms that shape genome architecture and evolution. Despite the ever-growing number of insect genomes sequenced to date, our understanding of the diversity and evolution of insect TEs remains poor., Results: Here, we present a standardized characterization and an order-level comparison of arthropod TE repertoires, encompassing 62 insect and 11 outgroup species. The insect TE repertoire contains TEs of almost every class previously described, and in some cases even TEs previously reported only from vertebrates and plants. Additionally, we identified a large fraction of unclassifiable TEs. We found high variation in TE content, ranging from less than 6% in the antarctic midge (Diptera), the honey bee and the turnip sawfly (Hymenoptera) to more than 58% in the malaria mosquito (Diptera) and the migratory locust (Orthoptera), and a possible relationship between the content and diversity of TEs and the genome size., Conclusion: While most insect orders exhibit a characteristic TE composition, we also observed intraordinal differences, e.g., in Diptera, Hymenoptera, and Hemiptera. Our findings shed light on common patterns and reveal lineage-specific differences in content and evolution of TEs in insects. We anticipate our study to provide the basis for future comparative research on the insect TE repertoire.

Background: Apoid wasps and bees (Apoidea) are an ecologically and morphologically diverse group of Hymenoptera, with some species of bees having evolved eusocial societies. Major problems for our understanding of the evolutionary history of Apoidea have been the difficulty to trace the phylogenetic origin and to reliably estimate the geological age of bees. To address these issues, we compiled a comprehensive phylogenomic dataset by simultaneously analyzing target DNA enrichment and transcriptomic sequence data, comprising 195 single-copy protein-coding genes and covering all major lineages of apoid wasps and bee families., Results: Our compiled data matrix comprised 284,607 nucleotide sites that we phylogenetically analyzed by applying a combination of domain- and codon-based partitioning schemes. The inferred results confirm the polyphyletic status of the former family "Crabronidae", which comprises nine major monophyletic lineages. We found the former subfamily Pemphredoninae to be polyphyletic, comprising three distantly related clades. One of them, Ammoplanina, constituted the sister group of bees in all our analyses. We estimate the origin of bees to be in the Early Cretaceous (ca. 128 million years ago), a time period during which angiosperms rapidly radiated. Finally, our phylogenetic analyses revealed that within the Apoidea, (eu)social societies evolved exclusively in a single clade that comprises pemphredonine and philanthine wasps as well as bees., Conclusion: By combining transcriptomic sequences with those obtained via target DNA enrichment, we were able to include an unprecedented large number of apoid wasps in a phylogenetic study for tracing the phylogenetic origin of bees. Our results confirm the polyphyletic nature of the former wasp family Crabonidae, which we here suggest splitting into eight families. Of these, the family Ammoplanidae possibly represents the extant sister lineage of bees. Species of Ammoplanidae are known to hunt thrips, of which some aggregate on flowers and feed on pollen. The specific biology of Ammoplanidae as predators indicates how the transition from a predatory to pollen-collecting life style could have taken place in the evolution of bees. This insight plus the finding that (eu)social societies evolved exclusively in a single subordinated lineage of apoid wasps provides new perspectives for future comparative studies.

It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA methylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes associated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/transcriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects.

Chalcidoidea are a megadiverse group of mostly parasitoid wasps of major ecological and economical importance that are omnipresent in almost all extant terrestrial habitats. The timing and pattern of chalcidoid diversification is so far poorly understood and has left many important questions on the evolutionary history of Chalcidoidea unanswered. In this study, we infer the early divergence events within Chalcidoidea and address the question of whether or not ancestral chalcidoids were small egg parasitoids. We also trace the evolution of some key traits: jumping ability, development of enlarged hind femora, and associations with figs. Our phylogenetic inference is based on the analysis of 3,239 single-copy genes across 48 chalcidoid wasps and outgroups representatives. We applied an innovative a posteriori evaluation approach to molecular clock-dating based on nine carefully validated fossils, resulting in the first molecular clock-based estimation of deep Chalcidoidea divergence times. Our results suggest a late Jurassic origin of Chalcidoidea, with a first divergence of morphologically and biologically distinct groups in the early to mid Cretaceous, between 129 and 81 million years ago (mya). Diversification of most extant lineages happened rapidly after the Cretaceous in the early Paleogene, between 75 and 53 mya. The inferred Chalcidoidea tree suggests a transition from ancestral minute egg parasitoids to larger-bodied parasitoids of other host stages during the early history of chalcidoid evolution. The ability to jump evolved independently at least three times, namely in Eupelmidae, Encyrtidae, and Tanaostigmatidae. Furthermore, the large-bodied strongly sclerotized species with enlarged hind femora in Chalcididae and Leucospidae are not closely related. Finally, the close association of some chalcidoid wasps with figs, either as pollinators, or as inquilines/gallers or as parasitoids, likely evolved at least twice independently: in the Eocene, giving rise to fig pollinators, and in the Oligocene or Miocene, resulting in non-pollinating fig-wasps, including gallers and parasitoids. The origins of very speciose lineages (e.g., Mymaridae, Eulophidae, Pteromalinae) are evenly spread across the period of chalcidoid evolution from early Cretaceous to the late Eocene. Several shifts in biology and morphology (e.g., in host exploitation, body shape and size, life history), each followed by rapid radiations, have likely enabled the evolutionary success of Chalcidoidea., (Copyright © 2017 Elsevier Inc. All rights reserved.)