An important question in cell biology is how DNA replication programs scale with genome size following polyploidization, an evolutionary process that increases DNA content without proportionally extending the cell cycle duration. In particular, comparative analyses across closely related polyploid species are lacking. Here, we developed an interspecies in vitro replication system using Xenopus laevis egg extracts and sperm nuclei from three closely related Xenopus species with distinct ploidy levels: diploid (Xenopus tropicalis), tetraploid (Xenopus laevis), and dodecaploid (Xenopus eysoole). Replication in diploid nuclei was faster than in tetraploid nuclei, consistent with higher fork density and speed, as expected. Counterintuitively, replication in dodecaploid nuclei was also accelerated relative to tetraploid nuclei, indicating that increased ploidy does not necessarily prolong S phase. Given the high conservation of replication genes, our results suggest that replication programs are dynamically tuned across polyploid species, revealing an unexpected flexibility in how eukaryotic cells adapt DNA replication to large-scale genome expansion.

Abstract Hammerhead ribozymes (HHRs) are small catalytic RNAs found across diverse life forms. In animal genomes, they can be encoded by genes organised in dispersed copies or in tandem genomic arrangements. These tandemly organised forms, known as Non-LTR retrozymes, were recently identified as a distinct group of non-autonomous retrotransposons, likely mobilised via a rolling-circle transposition mechanism and potentially involved in host transcriptome regulation. However, their evolutionary origins remain poorly understood. Here, we investigate the presence, genomic distribution, and possible origins of Non-LTR retrozymes across a broad range of vertebrate species. We find that these elements display a patchy phylogenetic distribution, notably absent from the Aves and Mammalia lineages. In species where they are present, retrozyme copy number, consensus length, and monomer proportion vary widely across species and retrozyme families, suggesting diverse amplification dynamics. Genomic mapping reveals a significant enrichment of Non-LTR retrozymes in intergenic regions and their exclusion from introns and exons, indicating selective pressure against genic insertion. Strikingly, the phylogenetic distribution of Non-LTR retrozymes coincides with that of Penelope-like elements (PLEs). Phylogenetic analysis further shows that the pLTR region of PLEs is closely related to Non-LTR retrozymes, supporting the hypothesis that Non-LTR retrozymes are non-autonomous derivatives of PLEs. Together, our findings shed new light on the evolutionary origin and genomic behaviour of Non-LTR retrozymes and underscore their potential regulatory roles in vertebrate genomes.

DNA replication in multicellular organisms follows a tightly regulated spatio-temporal program. Although the mechanisms underlying this replication program remain only partially understood, studies in model systems such as Xenopus laevis have highlighted the importance of titrating low-abundance replication factors. Whole-genome duplications as a consequence of polyploidization introduce additional layers of complexity, yet comparative analyses of replication programs across closely related polyploid species are scarce. Here, we developed an interspecies in vitro replication system using X. laevis egg extracts and sperm nuclei from three Xenopus species with varying ploidy: diploid (X. tropicalis), tetraploid (X. laevis), and dodecaploid (X. eysoole). Replication in diploid X. tropicalis was faster than in tetraploid X. laevis nuclei, due to higher fork density and speed. Surprisingly, dodecaploid X. eysoole replication was also accelerated compared to tetraploid nuclei, suggesting that higher ploidy does not necessarily extend S phase. Since replication genes are highly conserved between these species, these results imply dynamic tuning of replication programs across polyploid species and shed light on the evolutionary adaptability of DNA replication in response to genome duplication. ### Competing Interest Statement The authors have declared no competing interest. China Scholarship Council – Université Paris Saclay PhD fellowship, 202106760020

Sextonia rubra is a tropical tree endemic to the Guiana Shield and the Brazilian Amazon. Despite its renowned wood durability, it remains susceptible to degradation by white-rot fungi such as Trametes versicolor. To mitigate biotic stresses, plants can rely on their associated microbial communities, including endophytes, which play a crucial role in their defense mechanisms. In this study, we explored the cultivable microbiota of S. rubra, considering it a holobiont. Endophytic strains were isolated from the bark, sapwood and heartwood of S. rubra, and metabolome were extracted. We used a reverse chemical ecology approach to elucidate the mechanisms underlying these extracts' fungicidal activity. In this context, glutathione-S-transferases (GST), key detoxification enzymes of the lignivorous fungus T. versicolor, were chosen as targets. GST tests confirmed the presence of antifungal compounds in extracts from 13 of the 152 endophytes. Two isolates of Fusarium falciforme and one isolate of Fusarium graminearum were selected for co-culture experiments with T. versicolor. A comprehensive metabolic analysis of the confrontation zones using RPLC-ESI(+)-HRMS/MS and molecular networking revealed that the antifungal activity against T. versicolor was primarily mediated by cyclopeptides, and the observed contact inhibition was attributed to fusarins. These findings shed new light on the role of endophytic fungi in the chemical defense strategies of S. rubra, highlighting their potential as a source of bioactive compounds with antifungal properties.

BACKGROUND:Within eukaryotes, most horizontal transfer of genetic material involves mobile DNA sequences and such events are called horizontal transposable element transfer (HTT). Although thousands of HTT examples have been reported, the transfer mechanisms and their impacts on host genomes remain elusive. RESULTS:In this work, we carefully annotated three Helitron families within several Xenopus frog genomes. One of the Helitron family, Heli1Xen1, is recurrently involved in capturing and shuffling Xenopus laevis genes required in early embryonic development. Remarkably, we found that Heli1Xen1 is seemingly expressed in X. laevis and has produced multiple genomic polymorphisms within the X. laevis population. To identify the origin of Heli1Xen1, we searched its consensus sequence against available genome assemblies. We found highly similar copies in the genomes of another 13 vertebrate species from divergent vertebrate lineages, including reptiles, ray-finned fishes and amphibians. Further phylogenetic analysis provides evidence showing that Heli1Xen1 invaded these lineages via HTT quite recently, around 0.58-10.74 million years ago. CONCLUSIONS:The frequently Heli1Xen1-involved HTT events among reptiles, fishes and amphibians could provide insights into possible vectors for transfer, such as shared viruses across lineages. Furthermore, we propose that the Heli1Xen1 sequence could be an ideal candidate for studying the mechanism and genomic impact of Helitron transposition.

Helitron-like elements (HLEs) are widespread eukaryotic DNA transposons employing a rolling-circle transposition mechanism. Despite their prevalence in fungi, animals, and plant genomes, identifying Helitrons remains challenging. We introduce HELIANO, a software for annotating and classifying autonomous and non-autonomous Helitron and Helentron sequences from whole genomes. HELIANO outperforms existing tools in speed and accuracy, demonstrated through benchmarking and its application to complex genomes ( Xenopus tropicalis, Xenopus laevis, Oryza sativa ), revealing numerous newly identified Helitrons and Helentrons .In a comprehensive analysis of 404 eukaryote genomes, we found HLEs widely distributed across phyla, with exceptions in specific taxa. Helentrons were identified in numerous land plant species, and 20 protein domains were discovered integrated within specific autonomous HLE families. A global phylogenetic analysis confirmed the classification into main clades Helentron and Helitron , revealing nine subgroups, some enriched in particular taxa. The future use of HELIANO will contribute to the global analysis of TEs across genomes and enhance our understanding of this transposon superfamily.### Competing Interest StatementThe authors have declared no competing interest.

The mode of evolution of left-right asymmetries in the vertebrate habenulae remains largely unknown. Using a transcriptomic approach, we show that in a cartilaginous fish, the catshark Scyliorhinus canicula, habenulae exhibit marked asymmetries, in both their medial and lateral components. Comparisons across vertebrates suggest that those identified in lateral habenulae reflect an ancestral gnathostome trait, partially conserved in lampreys, and independently lost in tetrapods and neopterygians. Asymmetry formation involves distinct mechanisms in the catshark lateral and medial habenulae. Medial habenulae are submitted to a marked, asymmetric temporal regulation of neurogenesis, undetectable in their lateral counterparts. Conversely, asymmetry formation in lateral habenulae results from asymmetric choices of neuronal identity in post-mitotic progenitors, a regulation dependent on the repression of Wnt signaling by Nodal on the left. Based on comparisons with the mouse and the zebrafish, we propose that habenular asymmetry formation involves a recurrent developmental logic across vertebrates, which relies on conserved, temporally regulated genetic programs sequentially shaping choices of neuronal identity on both sides and asymmetrically modified by Wnt activity. The evolutionary origin of habenular asymmetries is elusive. Here they show morphological and molecular conservations indicative of an ancient origin in vertebrates and identify Wnt signaling as a core mechanism underlying their formation and diversification.

Helitron-like elements (HLEs) are widespread eukaryotic DNA transposons employing a rolling-circle transposition mechanism. Despite their prevalence in fungi, animals, and plant genomes, identifying Helitrons remains a formidable challenge. We introduce HELIANO, a software for annotating and classifying autonomous and non-autonomous HLE sequences from whole genomes. HELIANO overcomes several limitations of existing tools in speed and accuracy, demonstrated through benchmarking and its application to the complex genomes of frogs (Xenopus tropicalis and Xenopus laevis) and rice (Oryza sativa), where it uncovered numerous previously unidentified HLEs. In an extensive analysis of 404 eukaryote genomes, we found HLEs widely distributed across phyla, with exceptions in specific taxa. HELIANO’s application led to the discovery of numerous new HLEs in land plants and identified 20 protein domains captured by certain autonomous HLE families. A comprehensive phylogenetic analysis further classified HLEs into two primary clades, HLE1 and HLE2, and revealed nine subgroups, some of which are enriched within specific taxa. The future use of HELIANO promises to improve the global analysis of HLEs across genomes, significantly advancing our understanding of this fascinating transposon superfamily.

Glyphosate’s primary metabolite, aminomethylphosphonic acid (AMPA), is the most detected pollutant in surface waters. Recent studies have raised concerns about its toxicity, yet underlying mechanisms remain poorly understood. A disruption of the gut microbiome, which plays a crucial role in host health, could mediate most of the adverse effects. We investigated the impact of AMPA exposure on the gut microbiome of spined toad tadpoles ( Bufo spinosus ). We hypothesized that AMPA could alter the gut microbiota composition and that these effects could depend on the microbiota source. We exposed tadpoles to minute concentrations of AMPA and analyzed their faecal microbiota using 16S rRNA gene sequencing as a proxy of the gut microbiota. AMPA exposure decreased the gut bacterial biomass and affected the bacterial community composition of tadpole’s faeces. Furthermore, we observed interactions between AMPA exposure and maternal body condition on the Bacteroidota and Actinobacteriota phyla abundances. This suggests a maternal effect on early-life microbial colonizers that could influence the response of the gut microbiome to AMPA. These findings highlight the importance of considering the gut microbiome when studying the effects of environmental contaminants. Further research is needed to elucidate the long-term implications of this microbiome alteration for amphibian health.### Competing Interest StatementThe authors have declared no competing interest.

We gathered a collection of termite mutualistic strains from French Guiana to explore the metabolites of symbiotic microorganisms. Molecular networks reconstructed from a metabolomic analysis using LC–ESI–MS/MS methodology led us to identify two families of chlorinated polyketides, i.e. , azaphilones from Penicillium sclerotiorum and ilicicolins from Neonectria discophora . To define the biosynthetic pathways related to these two types of scaffolds, we used a whole genome sequencing approach followed by hybrid assembly from short and long reads. We found two biosynthetic gene clusters, including two FAD-dependent halogenases. To exploit the enzymatic promiscuity of the two identified FAD halogenases, we sought to biosynthesize novel halogenated metabolites. An OSMAC strategy was used and resulted in the production of brominated analogs of ilicicolins and azaphilones as well as iodinated analogs of azaphilones.

Background The Black Soldier Fly (BSF) Hermetia illucens is a cosmopolitan fly massively used by industrial companies to reduce biowaste and produce protein and fat for poultry and aquaculture feed. However, the natural history and the genetic diversity of the BSF are poorly known. Here, we present a comprehensive phylogeny and time tree based on a large dataset of complete mitochondrial genomes better to understand the evolution and timing of the BSF. Results In this study, we analyzed 677 CO1 sequences derived from samples found all over the five continents, leading us to discover 52 haplotypes, including ten major haplotypes. This worldwide cryptic genetic and genomic diversity is mirrored at a local scale in France, in which we found five major haplotypes sometimes in sympatry. Phylogenetic analyses of 60 complete mitochondrial genomes robustly resolved the phylogeny of the major BSF haplotypes. We estimate the separation events of the different haplotypes at more than 2 million years for the oldest branches characterizing the ancestral split between present North American lineages and the other highly diverse south-central American clades, possibly the following radiation beyond the isthmus of Panama northwards. Our data confirm that this North American lineage ultimately gave birth to almost all commercial BSF stocks that participated in the worldwide BSF dissemination through farm escapements. Conclusions Our data resolve the phylogenetic relationships between the major lineages and give insights into the BSF’s short and long-term evolution. Our results indicate that commercial BSF stock’s genetic and genomic diversity is very low. These results call for a better understanding of the genomic diversity of the BSF to unravel possible specific adaptations of the different lineages for industrial needs and to initiate the selection process.

Background Variation in locomotor capacity among animals often reflects adaptations to different environments. Despite evidence that physical performance is heritable, the molecular basis of locomotor performance and performance trade-offs remains poorly understood. In this study we identify the genes, signaling pathways, and regulatory processes possibly responsible for the trade-off between burst performance and endurance observed in Xenopus allofraseri , using a transcriptomic approach. Results We obtained a total of about 121 million paired-end reads from Illumina RNA sequencing and analyzed 218,541 transcripts obtained from a de novo assembly. We identified 109 transcripts with a significant differential expression between endurant and burst performant individuals (FDR ≤ 0.05 and logFC ≥2), and blast searches resulted in 103 protein-coding genes. We found major differences between endurant and burst-performant individuals in the expression of genes involved in the polymerization and ATPase activity of actin filaments, cellular trafficking, proteoglycans and extracellular proteins secreted, lipid metabolism, mitochondrial activity and regulators of signaling cascades. Remarkably, we revealed transcript isoforms of key genes with functions in metabolism, apoptosis, nuclear export and as a transcriptional corepressor, expressed in either burst-performant or endurant individuals. Lastly, we find two up-regulated transcripts in burst-performant individuals that correspond to the expression of myosin-binding protein C fast-type ( mybpc2 ). This suggests the presence of mybpc2 homoeologs and may have been favored by selection to permit fast and powerful locomotion. Conclusion These results suggest that the differential expression of genes belonging to the pathways of calcium signaling, endoplasmic reticulum stress responses and striated muscle contraction, in addition to the use of alternative splicing and effectors of cellular activity underlie locomotor performance trade-offs. Ultimately, our transcriptomic analysis offers new perspectives for future analyses of the role of single nucleotide variants, homoeology and alternative splicing in the evolution of locomotor performance trade-offs.

Background The Black Soldier Fly (BSF) Hermetia illucens is a cosmopolitan fly heavily used by industrial companies to reduce biowaste and produce protein and fat for poultry and aquaculture feed. However, the natural history and the genetic diversity of the BSF are poorly known. Results In this study, we analyzed 677 CO1 sequences derived from samples found all over the five continents, leading us to discover 52 haplotypes, including ten major haplotypes. We refined the definition of these haplotypes by sequencing 59 mitochondrial genomes. We could derive an estimate of the separation events of the different haplotypes at more than two million years for the oldest branches. This worldwide cryptic genetic and genomic diversity is mirrored at a local scale in France, in which we found five major haplotypes sometimes in sympatry. Conclusions Our data resolve the phylogenetic relationships between the major lineages and give insights into the dispersal and the numbers of BSF neo-introduction at global and local scales. Our results indicate that commercial BSF stock's genetic and genomic diversity is very low. In addition, this broodstock participates in disseminating the BSF in the wild. Taken together, these results call for a better understanding of the genomic diversity of the BSF to unravel possible specific adaptations of the different lineages for industrial needs and to initiate the selection process.

Background: The microorganism world living in amphibians is still largely under-represented and under-studied in the literature. Among anuran amphibians, African clawed frogs of the Xenopus genus stand as well-characterized models with an in-depth knowledge of their developmental biological processes including their metamorphosis. In this study, we analyzed the succession of microbial communities and their activities across diverse body habitats of Xenopus tropicalis using different approaches including flow cytometry and 16s rDNA gene metabarcoding. We also evaluated the metabolic capacity of the premetamorphic tadpoles gut microbiome using metagenomic and metatranscriptomic sequencing. Results: We analyzed the bacterial components of the Xenopus gut microbiota, the adult gut biogeography, the succession of communities during ontogeny, the impact of the alimentation in shaping the tadpoles gut bacterial communities and the transmission of skin and fecal bacteria to the eggs. We also identified the most active gut bacteria and their metabolic contribution to tadpole physiology including carbohydrate breakdown, nitrogen recycling, essential amino-acids and vitamin biosynthesis. Conclusions: We present a comprehensive new microbiome dataset of a laboratory amphibian model. Our data provide evidences that studies on the Xenopus tadpole model can shed light on the interactions between a vertebrate host and its microbiome. We interpret our findings in light of bile acids being key molecular components regulating the gut microbiome composition during amphibian development and metamorphosis. Further studies into the metabolic interactions between amphibian tadpoles and their microbiota during early development and metamorphosis should provide useful information on the evolution of host-microbiota interactions in vertebrates.

We describe a protocol to prepare a multiplexed mtDNA library from a blood sample for performing a long read sequencing of the mitochondrial genome. All steps are carefully described to get a high enrichment of mtDNA relative to total DNA extracted from the blood sample. The obtained mutiplexed library allows the production of long sequence mtDNA reads up to 16.5 kbp with a quality enabling variant-calling by using a portable sequencer (MinION, Oxford Nanopore Technologies).