Parthenogenetic Stick Insects Exhibit Signatures of Preservation in the Molecular Architecture of Male Reproduction.

After the loss of a trait, theory predicts that the molecular machinery underlying its phenotypic expression should decay. Yet, empirical evidence is contrasting. Here, we test the hypotheses that (i) the molecular ground plan of a lost trait could persist due to pleiotropic effects on other traits and (ii) that gene co-expression network architecture could constrain individual gene expression. Our testing ground has been the Bacillus stick insect species complex, which contains close relatives that are either bisexual or parthenogenetic. After the identification of genes expressed in male reproductive tissues in a bisexual species, we investigated their gene co-expression network structure in two parthenogenetic species. We found that gene co-expression within the male gonads was partially preserved in parthenogens. Furthermore, parthenogens did not show relaxed selection on genes upregulated in male gonads in the bisexual species. As these genes were mostly expressed in female gonads, this preservation could be driven by pleiotropic interactions and an ongoing role in female reproduction. Connectivity within the network also played a key role, with highly connected-and more pleiotropic-genes within male gonad also having a gonad-biased expression in parthenogens. Our findings provide novel insight into the mechanisms which could underlie the production of rare males in parthenogenetic lineages; more generally, they provide an example of the cryptic persistence of a lost trait molecular architecture, driven by gene pleiotropy on other traits and within their co-expression network.

Signatures of Extreme Longevity: A Perspective from Bivalve Molecular Evolution.

Among Metazoa, bivalves have the highest lifespan disparity, ranging from 1 to 500+ years, making them an exceptional testing ground to understand mechanisms underlying aging and the evolution of extended longevity. Nevertheless, comparative molecular evolution has been an overlooked approach in this instance. Here, we leveraged transcriptomic resources spanning 30 bivalve species to unravel the signatures of convergent molecular evolution in four long-lived species: Margaritifera margaritifera, Elliptio complanata, Lampsilis siliquoidea, and Arctica islandica (the latter represents the longest-lived noncolonial metazoan known so far). We applied a comprehensive approach-which included inference of convergent dN/dS, convergent positive selection, and convergent amino acid substitution-with a strong focus on the reduction of false positives. Genes with convergent evolution in long-lived bivalves show more physical and functional interactions to each other than expected, suggesting that they are biologically connected; this interaction network is enriched in genes for which a role in longevity has been experimentally supported in other species. This suggests that genes in the network are involved in extended longevity in bivalves and, consequently, that the mechanisms underlying extended longevity are-at least partially-shared across Metazoa. Although we believe that an integration of different genes and pathways is required for the extended longevity phenotype, we highlight the potential central roles of genes involved in cell proliferation control, translational machinery, and response to hypoxia, in lifespan extension.

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota.

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems.

Wood feeding and social living: Draft genome of the subterranean termite Reticulitermes lucifugus (Blattodea; Termitoidae).

Termites (Insecta, Blattodea, Termitoidae) are a widespread and diverse group of eusocial insects known for their ability to digest wood matter. Herein, we report the draft genome of the subterranean termite Reticulitermes lucifugus, an economically important species and among the most studied taxa with respect to eusocial organization and mating system. The final assembly (~813 Mb) covered up to 88% of the estimated genome size and, in agreement with the Asexual Queen Succession Mating System, it was found completely homozygous. We predicted 16,349 highly supported gene models and 42% of repetitive DNA content. Transposable elements of R. lucifugus show similar evolutionary dynamics compared to that of other termites, with two main peaks of activity localized at 25% and 8% of Kimura divergence driven by DNA, LINE and SINE elements. Gene family turnover analyses identified multiple instances of gene duplication associated with R. lucifugus diversification, with significant lineage-specific gene family expansions related to development, perception and nutrient metabolism pathways. Finally, we analysed P450 and odourant receptor gene repertoires in detail, highlighting the large diversity and dynamical evolutionary history of these proteins in the R. lucifugus genome. This newly assembled genome will provide a valuable resource for further understanding the molecular basis of termites biology as well as for pest control.

Gene transcriptional profiles in gonads of Bacillus taxa (Phasmida) with different cytological mechanisms of automictic parthenogenesis.

The evolution of automixis - i.e., meiotic parthenogenesis - requires several features, including ploidy restoration after meiosis and maintenance of fertility. Characterizing the relative contribution of novel versus pre-existing genes and the similarities in their expression and sequence evolution is fundamental to understand the evolution of reproductive novelties. Here we identify gonads-biased genes in two Bacillus automictic stick-insects and compare their expression profile and sequence evolution with a bisexual congeneric species. The two parthenogens restore ploidy through different cytological mechanisms: in Bacillus atticus, nuclei derived from the first meiotic division fuse to restore a diploid egg nucleus, while in Bacillus rossius, diploidization occurs in some cells of the haploid blastula through anaphase restitution. Parthenogens' gonads transcriptional program is found to be largely assembled from genes that were already present before the establishment of automixis. The three species transcriptional profiles largely reflect their phyletic relationships, yet we identify a shared core of genes with gonad-biased patterns of expression in parthenogens which are either male gonads-biased in the sexual species or are not differentially expressed there. At the sequence level, just a handful of gonads-biased genes were inferred to have undergone instances of positive selection exclusively in the parthenogen species. This work is the first to explore the molecular underpinnings of automixis in a comparative framework: it delineates how reproductive novelties can be sustained by genes whose origin precedes the establishment of the novelty itself and shows that different meiotic mechanisms of reproduction can be associated with a shared molecular ground plan.

Curation of a reference database of COI sequences for insect identification through DNA metabarcoding: COins.

DNA metabarcoding is a widespread approach for the molecular identification of organisms. While the associated wet-lab and data processing procedures are well established and highly efficient, the reference databases for taxonomic assignment can be implemented to improve the accuracy of identifications. Insects are among the organisms for which DNA-based identification is most commonly used; yet, a DNA-metabarcoding reference database specifically curated for their species identification using software requiring local databases is lacking. Here, we present COins, a database of 5' region cytochrome c oxidase subunit I sequences (COI-5P) of insects that includes over 532 000 representative sequences of >106 000 species specifically formatted for the QIIME2 software platform. Through a combination of automated and manually curated steps, we developed this database starting from all COI sequences available in the Barcode of Life Data System for insects, focusing on sequences that comply with several standards, including a species-level identification. COins was validated on previously published DNA-metabarcoding sequences data (bulk samples from Malaise traps) and its efficiency compared with other publicly available reference databases (not specific for insects). COins can allow an increase of up to 30% of species-level identifications and thus can represent a valuable resource for the taxonomic assignment of insects' DNA-metabarcoding data, especially when species-level identification is needed https://doi.org/10.6084/m9.figshare.19130465.v1.

Macroevolutionary Analyses Provide New Evidence of Phasmid Wings Evolution as a Reversible Process.

The concept that complex ancestral traits can never be recovered after their loss is still widely accepted, despite phylogenetic and molecular approaches suggest instances where phenotypes may have been lost throughout the evolutionary history of a clade and subsequently reverted back in derived lineages. One of the first and most notable examples of such a process is wing evolution in phasmids; this polyneopteran order of insects, which comprises stick and leaf insects, has played a central role in initiating a long-standing debate on the topic. In this study, a novel and comprehensive time tree including over 300 Phasmatodea species is used as a framework for investigating wing evolutionary patterns in the clade. Despite accounting for several possible biases and sources of uncertainty, macroevolutionary analyses consistently revealed multiple reversals to winged states taking place after their loss, and reversibility is coupled with higher species diversification rates. Our findings support a loss of or reduction in wings that occurred in the lineage leading to the extant phasmid most recent common ancestor, and brachyptery is inferred to be an unstable state unless co-opted for nonaerodynamic adaptations. We also explored how different assumptions of wing reversals probability could impact their inference: we found that until reversals are assumed to be over 30 times more unlikely than losses, they are consistently inferred despite uncertainty in tree and model parameters. Our findings demonstrate that wing evolution is a reversible and dynamic process in phasmids and contribute to our understanding of complex trait evolution. [Dollo's law; Phasmatodea; phylogenetic comparative methods; polyneoptera; reversals; wing.].

Comparative genomics of tadpole shrimps (Crustacea, Branchiopoda, Notostraca): Dynamic genome evolution against the backdrop of morphological stasis.

This analysis presents five genome assemblies of four Notostraca taxa. Notostraca origin dates to the Permian/Upper Devonian and the extant forms show a striking morphological similarity to fossil taxa. The comparison of sequenced genomes with other Branchiopoda genomes shows that, despite the morphological stasis, Notostraca share a dynamic genome evolution with high turnover for gene families' expansion/contraction and a transposable elements content comparable to other branchiopods. While Notostraca substitutions rate appears similar or lower in comparison to other branchiopods, a subset of genes shows a faster evolutionary pace, highlighting the difficulty of generalizing about genomic stasis versus dynamism. Moreover, we found that the variation of Triops cancriformis transposable elements content appeared linked to reproductive strategies, in line with theoretical expectations. Overall, besides providing new genomic resources for the study of these organisms, which appear relevant for their ecology and evolution, we also confirmed the decoupling of morphological and molecular evolution.

BASE: A novel workflow to integrate nonubiquitous genes in comparative genomics analyses for selection.

Inferring the selective forces that orthologous genes underwent across different lineages can help us understand the evolutionary processes that have shaped their extant diversity and the phenotypes they underlie. The most widespread metric to estimate the selection regimes of coding genes-across sites and phylogenies-is the ratio of nonsynonymous to synonymous substitutions (dN/dS, also known as ω). Nowadays, modern sequencing technologies and the large amount of already available sequence data allow the retrieval of thousands of orthologous genes across large numbers of species. Nonetheless, the tools available to explore selection regimes are not designed to automatically process all genes, and their practical usage is often restricted to the single-copy ones which are found across all species considered (i.e., ubiquitous genes). This approach limits the scale of the analysis to a fraction of single-copy genes, which can be as low as an order of magnitude in respect to those which are not consistently found in all species considered (i.e., nonubiquitous genes). Here, we present a workflow named BASE that-leveraging the CodeML framework-eases the inference and interpretation of gene selection regimes in the context of comparative genomics. Although a number of bioinformatics tools have already been developed to facilitate this kind of analyses, BASE is the first to be specifically designed to allow the integration of nonubiquitous genes in a straightforward and reproducible manner. The workflow-along with all relevant documentation-is available at github.com/for-giobbe/BASE.

Phylomitogenomics provides new perspectives on the Euphasmatodea radiation (Insecta: Phasmatodea).

Phasmatodea species diversity lies almost entirely within its suborder Euphasmatodea, which exhibits a pantropical distribution and is considered to derive from a recent and rapid evolutionary radiation. To shed light on Euphasmatodea origins and diversification, we assembled the mitogenomes of 17 species from transcriptomic sequencing data and analysed them along with 22 already available Phasmatodea mitogenomes and 33 mitogenomes representing most of the Polyneoptera lineages. Maximum Likelihood and Bayesian Inference approaches retrieved consistent topologies, both showing the widespread conflict between phylogenetic approaches and traditional systematics. We performed a divergence time analysis leveraging ten fossil specimens representative of most polyneopteran lineages: the time tree obtained supports an older radiation of the clade with respect to previous hypotheses. Euphasmatodea diversification is inferred to have started ~ 187 million years ago, suggesting that the Triassic-Jurassic mass extinction and the breakup of Pangea could have contributed to the process. We also investigated Euphasmatodea mitogenomes patterns of dN, dS and dN/dS ratio throughout our time-tree, trying to characterize the selective regime which may have shaped the clade evolution.

Complete mitochondrial genomes from transcriptomes: assessing pros and cons of data mining for assembling new mitogenomes.

Thousands of eukaryotes transcriptomes have been generated, mainly to investigate nuclear genes expression, and the amount of available data is constantly increasing. A neglected but promising use of this large amount of data is to assemble organelle genomes. To assess the reliability of this approach, we attempted to reconstruct complete mitochondrial genomes from RNA-Seq experiments of Reticulitermes termite species, for which transcriptomes and conspecific mitogenomes are available. We successfully assembled complete molecules, although a few gaps corresponding to tRNAs had to be filled manually. We also reconstructed, for the first time, the mitogenome of Reticulitermes banyulensis. The accuracy and completeness of mitogenomes reconstruction appeared independent from transcriptome size, read length and sequencing design (single/paired end), and using reference genomes from congeneric or intra-familial taxa did not significantly affect the assembly. Transcriptome-derived mitogenomes were found highly similar to the conspecific ones obtained from genome sequencing (nucleotide divergence ranging from 0% to 3.5%) and yielded a congruent phylogenetic tree. Reads from contaminants and nuclear transcripts, although slowing down the process, did not result in chimeric sequence reconstruction. We suggest that the described approach has the potential to increase the number of available mitogenomes by exploiting the rapidly increasing number of transcriptomes.

Mitochondrial genome diversity and evolution in Branchiopoda (Crustacea).

BACKGROUND: The crustacean class Branchiopoda includes fairy shrimps, clam shrimps, tadpole shrimps, and water fleas. Branchiopods, which are well known for their great variety of reproductive strategies, date back to the Cambrian and extant taxa can be mainly found in freshwater habitats, also including ephemeral ponds. Mitochondrial genomes of the notostracan taxa Lepidurus apus lubbocki (Italy), L. arcticus (Iceland) and Triops cancriformis (an Italian and a Spanish population) are here characterized for the first time and analyzed together with available branchiopod mitogenomes. RESULTS: Overall, branchiopod mitogenomes share the basic structure congruent with the ancestral Pancrustacea model. On the other hand, rearrangements involving tRNAs and the control region are observed among analyzed taxa. Remarkably, an unassigned region in the L. apus lubbocki mitogenome showed a chimeric structure, likely resulting from a non-homologous recombination event between the two flanking trnC and trnY genes. Notably, Anostraca and Onychocaudata mitogenomes showed increased GC content compared to both Notostraca and the common ancestor, and a significantly higher substitution rate, which does not correlate with selective pressures, as suggested by dN/dS values. CONCLUSIONS: Branchiopod mitogenomes appear rather well-conserved, although gene rearrangements have occurred. For the first time, it is reported a putative non-homologous recombination event involving a mitogenome, which produced a pseudogenic tRNA sequence. In addition, in line with data in the literature, we explain the higher substitution rate of Anostraca and Onychocaudata with the inferred GC substitution bias that occurred during their evolution.

Patterns of CRISPR/Cas9 activity in plants, animals and microbes.

The CRISPR/Cas9 system and related RNA-guided endonucleases can introduce double-strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on-target mutations), the targeting accuracy (likelihood of off-target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species-dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome-editing tools.