Genome Biology and Evolution

Gene duplication is a major driver of genomic innovation, but whether paralog accumulation scales uniformly with increasing genome size across the domain Archaea remains unclear. This study presents a domain-wide analysis of 1,829 archaeal genomes and their predicted proteomes to evaluate protein family expansions, their distribution, and lineage-specific patterns of gene duplication. Protein family expansions were classified as recent (90% sequence identity), intermediate (70%), or old (40%) duplications based on sequence-identity thresholds within each family. The findings show that gene duplication content is decoupled from overall proteome size at high sequence-identity levels, indicating that recent paralog retention does not correlate with proteome expansion. Archaeal lineages exhibit contrasting gene duplication profiles. All 1,829 genomes contained paralogs at the 40% sequence identity threshold, indicating the widespread presence of ancient or divergent duplication events. In contrast, paralogs detected at the 90% identity threshold, showed substantial variation across lineages. Their proportion reached up to 16.6% in groups such as Halobacteria, whereas only 74 genomes (4.04%) completely lacked paralogs at this threshold. Highly duplicated families are enriched for membrane transport systems (ABC/MFS), central metabolic enzymes (e.g., acetyl-CoA synthetase), a high proportion of uncharacterized functions, and mobile elements, predominantly transposases. Notably, we identified 314 transposase protein families in Archaea, distributed in 161 unique families in Archaea and 153 families shared with Bacteria, suggesting mechanisms of interdomain gene transfer. Overall, paralog accumulation in Archaea is a lineage-specific phenomenon shaped by targeted functional duplications and mobile element activity, rather than a simple consequence of proteome growth.

GC-biased gene conversion (gBGC) is a widespread recombination-associated process that favours the fixation of GC alleles independently of their fitness. In coding regions, it can oppose purifying selection and favour the accumulation of deleterious AT to GC mutations. In mammals where recombination hotspots are predominantly determined by PRDM9, the deleterious effect of gBGC is expected to be episodic due to the rapid turnover of hotspots. The deleterious impact of gBGC episodes on proteins has been well documented, particularly in rodents and primates. However, the evolutionary response of the affected proteins after the episodes end has been scarcely investigated. Theory predicts that a decline in gBGC intensity should trigger a compensatory response that enables previous damage to be repaired. Recently, we reported evidence for pervasive compensatory evolution following gBGC episodes in Hydromyini rodents. Here, we aimed to investigate whether this post- gBGC compensation is widespread among mammals in which gBGC is episodic. Using a phylogenetic approach, we analysed substitution patterns in coding exons from five mammalian taxa spanning a range of effective population size (Ne): Murini, Rattini, Cetacea, Cercopithecidae and Hominoidae. We detected thousands of gBGC episodes, characterised by an increased rate of non-synonymous AT to GC substitutions. We found evidence for compensatory evolution in non-primate taxa, and a lack of signal in Primates. Although the influence of Ne remains unclear, our study highlights both the evolutionary significance of post-gBGC compensation and the methodological challenges in detecting it.

A significant fraction of archaeal genomes have their genomic content distributed across two or more DNA molecules, yet the characteristics and evolution of these different replicons remain poorly understood. Here, we investigated the genomic and functional organization of secondary replicons in Halobacteriota using 211 complete genomes, comprising 766 replicons, including 211 primary chromosomes (chr1), and 555 secondary replicons. Comparative analyses of genomic features, including replicon size, GC content, and codon usage, revealed a continuous distribution of secondary replicons from plasmid-like to chromosome-like characteristics. Larger secondary replicons showed progressively more chromosome-like genomic and functional properties, including greater protein-family overlap with chr1 and a higher proportion of conserved gene families. At the phylum level, functional profiles substantially overlapped among replicon types, providing further support for a continuum rather than discrete functional classes. Larger replicons also showed increased protein-family duplication, while pangenome analysis revealed a greater proportion of conserved gene families, with accessory families most abundant at intermediate sizes and rare and singleton families declining with increasing replicon size. Together, these results support a model in which secondary replicons in Halobacteriota span a genomic and functional continuum from plasmid-like to chromosome-like states , with increasing size associated with greater conservation, gene duplication and progressively more chromosome-like gene content and functional profile.

Forms I&prime, I&Prime, and I RuBisCOs were recently identified in bacterial metagenome-assembled genomes (MAGs) and are significant for evolutionarily bridging form I, which is responsible for the major part of carbon fixation on Earth, and other forms. Pioneering studies focused mainly on the biochemical and structural characteristics of "form I-related" RuBisCOs and deduced the evolution of this protein family over phylogenetic relationships among form I and form I-related sequences. We here surveyed form I-related sequences in public databases and conducted phylogenetic analyses incorporating the updated diversity of form I-related RuBisCOs. This study yielded two major outcomes regarding RuBisCO evolution. First, the analyses presented here suggest uncertainties overlooked in the previously published studies. It appeared to be difficult to conclude (i) the phylogenetic relationship among forms I, I&prime, and I&Prime, (ii) the monophyly of form I&prime, or (iii) the monophyly of form I. Second, we identified a gene encoding form I RuBisCO in an archaeal MAG, and this protein is likely involved in the pentose bisphosphate pathway. The results presented here suggest that the evolutionary route leading to form I in RuBisCO evolution remains unsettled.

Tandemly repeated sequences are noncoding, highly variable, yet integral components of eukaryotic genomes. Empirical research has so far largely focused on a few well-characterized, functional tandem repeats (TRs) or documented the broad diversity and rapid evolution of these repeats without explicitly testing the forces driving their evolution. By characterizing TR abundance and TR sequence composition within and between chromosomes and species in the stick insect genus Timema, we uncover that TR loads are enriched in genomic regions of high recombination but tend to be reduced in species with higher effective population sizes. These results suggest that there is a mutational effect of recombination that generates increased TR loads and that the bulk of TRs experience weak selection, leading to their rapid turnover over short evolutionary timescales. Finally, comparisons of centromere and non-centromere TRs reveal that the rapid divergence of centromeres is best explained by the combination of the fast-evolving nature of TRs per se and centromere-specific evolutionary forces. Overall, this study provides the first in-depth investigation of genome-wide TR evolution, while integrating the effects of recombination, genetic drift, and centromeric position along the genome.

Clownfishes are a complex of 28 species that rapidly diversified after acquiring mutualism with host sea anemones around 15 million years ago. The genomic mechanisms behind this radiation are only beginning to emerge, and structural variants (SVs) have received little attention. Previous phylogenomic studies suggested two large inversions on chromosome 18, but their distribution across the clownfish phylogeny and their potential effects have not been formally explored. SVs can disrupt gene function and regulation or alter gene dosage, and they have been shown to play a central role in adaptive evolution and diversification across many taxa. Here, we characterized SVs across the clownfish radiation using long-read PacBio sequencing, generating high-quality genome assemblies for sixteen species spanning the main lineages of the genus. We identified and catalogued nearly 130,000 SVs spanning close to 570 Mb. Short insertions and deletions were the most numerous, but large inversions accounted for most of the affected sequence. No SV was consistently associated with host specialization. We nevertheless confirmed a single inversion of approximately 17 Mb on chromosome 18, clarifying earlier reports of two separate rearrangements at this locus, and identified an additional inversion on chromosome 9 whose distribution suggests a history of hybridization. This study provides the first genome-wide characterization of structural variation in clownfishes, revealing extensive SVs despite the group's rapid and recent radiation. It also delivers new, high-quality genomic resources that will support future research into this iconic group of coral reef fishes and their adaptation to sea anemones.

We present draft genome assemblies for two poneroid ant species with distinctive life history traits: Platythyrea mocquerysi Emery, 1899 (Ponerinae) and Stigmatomma oregonense Wheeler, 1915 (Amblyoponinae). Platythyrea mocquerysi is a Malagasy ant in which larvae produce secretions from a ventral tubercle that are consumed by adults, representing an unusual form of larva-to-adult trophallaxis. Stigmatomma oregonense, a North American "dracula ant," feeds non-lethally on larval haemolymph. Using PacBio HiFi long-read sequencing, we assembled the P. mocquerysi genome (187 Mb, 307 scaffolds, N50 = 5.26 Mb, BUSCO 99.2% complete) and the S. oregonense genome (262 Mb, 1,189 scaffolds, N50 = 0.84 Mb, BUSCO 98.3% complete). These genomes encode 10,860 and 12,354 protein-coding genes, respectively. Repeat content is higher in S. oregonense than in P. mocquerysi, 54.3% against a lower bound of 15.7%. Most of the difference is in unclassified repeats, which we show to be diverged transposable elements. Comparative analysis with two additional poneroid genomes from closely related species reveals that genome size variation across poneroid ants is largely explained by transposable element dynamics, with notable expansions in several gene families including carboxylesterases, serine proteases, and lipid metabolism genes in Amblyoponinae. These genomes provide essential resources for investigating the molecular basis of unconventional brood management strategies in ants.

The allorecognition system of the hydroid Hydractinia symbiolongicarpus plays an essential role in mediating self vs. non-self recognition among cnidarian colonies, analogous to the role of the major histocompatibility complex (MHC) in bilaterian immune systems. Both complexes have contiguous stretches of similar, highly duplicated genes, suggesting a homologous relationship. Protein structural prediction methods such as AlphaFold2 also indicate structural similarity between the proteins encoded within the Hydractinia ARC and the MHC. We have determined the structure of the ARC in the related hydrozoan Podocoryna americana to determine the degree of conservation of the cnidarian allorecognition complex. Protein structural prediction methods have revealed a number of candidate allorecognition proteins that form a long contiguous complex. Phylogenies suggest homology between cnidarian allorecognition genes, as well as syntenic conservation of this gene complex across hydrozoans. Ongoing comparative genomic analyses across bilaterian species aim to clarify the relationship between the ARC and the MHC, thereby informing our understanding of the evolutionary origins of bilaterian adaptive immunity.

While studies of molecular convergence typically focus on species that independently evolved similar phenotypes, similar questions can be asked among populations of different species adapting to the same environment. Here, we study the Baltic Sea environmental gradient and two co-distributed, ecologically similar, but distantly related pipefish species, the broadnosed pipefish (Syngnathus typhle) and the straightnose pipefish (Nerophis ophidion), to test whether independent colonization of the Baltic Sea led to similar population structure and caused convergence at different genomic levels. Using a new diploid genome for S. typhle and whole-genome resequencing data (N=99 for S. typhle; N=81 for N. ophidion) across seven locations from France to Finland, we identified stronger population structure in S. typhle than in N. ophidion, while both species showed differentiation between North Atlantic, Danish Straits, and Baltic Sea populations. Genetic diversity was lower in Baltic populations, particularly in S. typhle. Cross-species comparisons of Baltic populations revealed contrasting chromosome-level patterns of genomic differentiation, with differentiation spread across the genome in S. typhle but concentrated in specific chromosomal regions in N. ophidion. Nonetheless, we identified multiple orthologous genes and SNPs showing convergent differentiation in the Baltic populations of both species, including genes related to metabolism, immunity, and regulatory functions. Overall, we identified molecular convergence at the gene and nucleotide level between Baltic populations of distantly related pipefish species, while convergence was limited at broader genomic scales. These findings suggest that similar environmental pressures can repeatedly target specific genetic elements even when genomic backgrounds and population structures differ.

Universal Single-Copy Orthologs (USCOs) are taxonomically-defined gene sets originally proposed to evaluate completeness in genome assemblies. Due to their single- or low-copy nature, they have also been proposed as ideal markers for phylogenomics. Here, we test this assumption using two vertebrate datasets, mammals and ray-finned fish, and their corresponding USCO reference marker sets, actinopterygii_odb10 and mammalia_odb10. Our results show that although most USCO markers are single-copy, 85% and 91% respectively, empirical analyses using a large number of taxa are able to identify a non-negligible proportion of paralogs in the actinopterygii_odb10 and mammalia_odb10 BUSCO gene sets. Gene phylogenies show that several of these paralogs derive from ancient whole-genome duplication events, particularly the teleost-specific whole-genome duplication, and also more recent duplications such as those in salmonids and cyprinids. Paralogs do not reflect the organismal phylogeny but rather the history of gene duplications, so retaining paralogs in phylogenomic datasets can lead to inaccurate phylogenomic hypotheses. To test the effect of ignoring paralogy in BUSCO gene sets, we analyze the mammal and ray-finned fish datasets using concatenated maximum likelihood and summary coalescent methods by randomly choosing one paralog per locus if these are present. The resulting phylogenomic trees are overall consistent with the reference phylogenies, although small discrepancies were found. Phylogeny-guided paralogy removal also changes the sequences retained for many loci, but whether these changes alter the inferred species trees remains to be tested.

The twisted-wing parasites (order Strepsiptera) are well-known for their unusual life histories as obligate endoparasites of a wide variety of other insect orders. Here, we present the first high-quality genome assembly of a leafhopper-parasitizing strepsipteran species. We sequenced the genome of Elenchus koebelei using PacBio HiFi technology, yielding a primary assembly of 63.7Mb in 96 contigs, with an N50 of 1.60Mb and a BUSCO completeness score of 82.3%. In total, we recovered 8,167 protein-coding gene annotations, and 34.2% of the genome annotated as repeats. For the first time, we compare the genome content of three strepsipteran species and find that they are remarkably consistent in broad structure, with approximately 600 insect universal single-copy orthologs that appear to be completely missing from the order. These similarities imply a conserved genomic blueprint for strepsipteran parasitic strategy and/or differences that must be investigated at the post-translational level.

{sigma}-factors are dissociable subunits of RNA polymerase (RNAP), forming the basal regulatory machinery for bacterial transcription. The minimal {sigma}-factors contain two domains (called {sigma}2 or {sigma}4), both of which bind RNAP as well as the promoter DNA through their helix-turn-helix motif. Housekeeping {sigma}-factors contain four domains. We identify proteins containing at least one of the four {sigma}-factor domains in bacterial and bacteriophage genomes; we also show that proteins containing either {sigma}2 or {sigma}4, which are not defined as {sigma}-factors, are commonly found in both sets of organisms. Using phylogenetic analysis of these two domains, we suggest that the highly conserved major housekeeping {sigma}-factor, with its complex domain architecture, represents a late-evolving monophyletic clade, whereas single-domain proteins and minimal {sigma}-factor architectures comprising {sigma}2 and {sigma}4 might be ancestral. Thus, {sigma}-factors probably evolved from single-domain DNA-binding transcription factors by domain accretion.

Two rounds of whole-genome duplication (2R-WGD) in the vertebrate lineage are thought to have driven gene diversification. Mammals have four genes encoding five nebulin superfamily proteins (LASP1, LASP2/NEBL, NEB, and NRAP) hypothesised to have arisen from invertebrate LASP via WGD. Here, we examined the evolutionary fate of the nebulin superfamily after an additional WGD, teleost-specific genome duplication (TGD). Genome and transcriptome analysis of zebrafish (Danio rerio) and medaka (Oryzias latipes), of which the complete genomes were recently determined, showed only lasp family genes retained duplicate copies. Zebrafish retained two LASP2/NEBL-derived ohnologs; one (LASP2L) had been misidentified as a nebulette gene in the Zebrafish Information Network (ZFIN) despite lacking nebulette-specific exons. Another (LASP2/NEBL) is newly identified in this study because it lies in a newly determined region of the latest genome assembly. Contrastingly, medaka retained two LASP1 ohnologs and one LASP2/NEBL. The duplicated lasp family proteins showed overlapping and specific tissue expression patterns. We confirmed all three zebrafish lasp proteins bound actin and co-localized with actin-rich structures when ectopically expressed in cultured fibroblasts. This supports that TGD-derived lasp duplicates underwent lineage-specific retention and expression divergence in teleosts whilst conserving core actin-binding properties.

Phenotypic plasticity is a common adaptation to heterogeneous environments. While it is well documented that trait plasticity can rapidly evolve, little is known about the developmental mechanisms that underlie variation in plasticity. Here, we compare diverged populations of the common buckeye butterfly (Junonia coenia): one population with canalized wing patterns that are insensitive to environmental conditions and another population that has distinctive seasonal color patterns. In common garden trials we were surprised to find dramatically elevated levels of transcriptional and chromatin accessibility plasticity during wing development of the canalized population compared to the seasonally plastic population. Further, using machine learning analysis of accessible chromatin sequences, we observed a broad heterochronic shift in motifs enriched in plastic putative regulatory elements to earlier stages of development in the canalized population, which we speculate may cause phenotype-related gene expression to move outside a wing color critical period. Together, our data lead us to propose a model where cryptic genetic processes are masked by a shift in chromatin remodeling, which in turn could produce a capacitor-like effect to maintain developmental genetic variation in the canalized population.

Conserved synteny enables the reconstruction of ancestral chromosome organization across deeply diverged lineages, as demonstrated across metazoans. In Spiralia, comparative genomic analyses have suggested an ancestral karyotype comprising 20 chromosomes. We report high-quality genome assemblies for two Lima species (Lima caribaea and Lima lima) with 20 chromosomes and show that their chromosome organization matches this ancestral state, including four previously described fusion-with-mixing events. Interestingly, other Limidae genus did not retain the ancestral karyotype: they have undergone subsequent lineage-specific fusions. The conserved organization of Lima chromosomes provides a rare genomic anchor for reconstructing ancestral chromosome organization and lineage-specific chromosome evolution across Spiralia and, more broadly, Metazoa.

Heterogeneous genomic landscapes of differentiation can reveal evolutionary processes acting over different timescales. Whereas genetic structure in the European flat oyster, Ostrea edulis, is mainly shaped by geography, previous work suggested parallel differentiation between western and eastern Mediterranean populations and between Atlantic and North Sea populations, resulting in genetic convergence between range margin populations in some genomic regions. Here, we analyzed 27 oysters from four locations using moderate-coverage (~20X) short-read whole-genome sequencing. We first examined three known chromosomal inversions and found evidence that they predate divergence with the sister species O. chilensis. We then scanned the genome for parallel differentiation at the individual-SNP rather than sliding-window scale using the product of allele-frequency differences across the two geographic contrasts. Two of the three inversions were detected, but we also identified 12 small non-inverted genomic regions showing even stronger signals of parallel differentiation. These 'islands of parallelism' typically overlap one or a few genes, and most showed elevated dXY consistent with long-term barriers to gene flow. Two islands overlap nuclear ATP synthase subunits, while mitochondrial ATP8 shows a convergent amino-acid change, suggesting possible nuclear-nuclear and mitonuclear incompatibilities. Finally, demographic inference with heterogeneous gene flow supported post-glacial secondary contact and a lineage-based rather than geography-based population topology. Our results suggest that the genomes of O. edulis lineages have been largely porous to gene flow upon secondary contact, except for chromosomal inversions and small genomic islands. These regions likely act as barrier loci, preserving a signal of a shared evolutionary history among marginal populations that has otherwise been erased by rampant gene flow.

Eucalyptus recurva (Mongarlowe Mallee) is Critically Endangered, with only six known adult individuals persisting across two sites in the Southern Tablelands of New South Wales, Australia. Its extreme rarity, uniquely long lifespan, and limited reproductive output make it a priority for conservation genomics. Here we report the first genome assembly of E. recurva: a haplotype-resolved, gapless, telomere-to-telomere (T2T) assembly produced from Oxford Nanopore Technologies (ONT) long-read sequencing. Both haplotypes span 11 chromosomes (consistent with the conserved Eucalyptus karyotype of 2n = 22), with assembly sizes of 521.1 Mb (Hap 1) and 501.5 Mb (Hap 2), BUSCO completeness >99.6%, and quality values of >QV 62. Comparative analyses place E. recurva within section Maidenaria as sister to Eucalyptus viminalis and reveal high synteny between the two species. Inter-haplotype comparison identified 3.6 million SNPs and modest structural variation, suggesting that, despite extreme demographic bottlenecking, E. recurva retains meaningful genomic heterozygosity. We also characterise the chloroplast genome, which exhibits heteroplasmy, and report an apparently bipartite mitochondrial genome. This reference genome provides an essential resource for conservation management, population genetics, and the study of eucalypt genome evolution.

Whole-genome duplications (WGDs) have long been proposed as evolutionary facilitators of biological complexity and diversification. Ancient WGDs occurred deep in the evolutionary history of both plants and vertebrates, but appear to be rare in invertebrates. However, it is unclear whether this reflects genuine scarcity or inadequate detection methodologies, and current methods may be insufficient to detect very old WGDs where extensive gene loss has followed. Here, we used a combination of paralogous and orthologous synteny-based methods incorporating bilaterian ancestral linkage groups (ALGs) to search for genomic signatures of WGD in members of the invertebrate phylum Bryozoa. This revealed strong evidence for an ancient WGD in the freshwater bryozoan Cristatella mucedo, Class Phylactolaemata, with duplicated paralogous regions across every chromosome. Only ~10% of duplicated genes are retained, including a Hox cluster duplication. All eight genomes from the bryozoan class Gymnolaemata also show signatures of ancient WGD in the form of 2:1 ratios in orthologous synteny comparisons, which remain detectable despite only ~5% duplicate retention. Gene trees support a scenario in which all extant bryozoans share an ancient WGD and much of the genome rediploidised independently in the two clades. Duplicates retained after bryozoan WGD are enriched in cilia genes and are preferentially expressed in the lophophore, the tentacular feeding organ covered in highly specialised cilia. We argue that cryptic WGD in bryozoan evolution facilitated adaptation to a sessile, filter-feeding lifestyle, and propose that incorporation of ALGs adds to the power of synteny-based WGD-detection methods.

Regulatory variation is widely recognized as a driver of evolution within and between species, including in the primate lineage. Evidence for potentially adaptive regulatory evolution in primates comes from both sequence-based tests for selection and cross-species comparisons of gene expression. However, comparative studies of gene expression have tended to focus on baseline cellular states, despite growing evidence that context-dependent gene regulation is important. Here, we investigate the evolution of gene expression in lymphoblastoid cell lines (LCLs) from 6 primate lineages (humans, chimpanzees, bonobos, western gorillas, Sumatran orangutans, and rhesus macaques) using data from both control and 5 perturbed cellular conditions (n=22-25 individuals per condition). Analyzing baseline expression data, we find that genes involved in immunity often exhibit lineage-specific transcriptional shifts, and that these genes occur near regions with lineage-specific sequence acceleration and lineage-specific epigenomic states. However, the subset of genes that respond to in vitro perturbations have evolved at significantly higher rates than environmentally-insensitive genes, a finding we replicate in an independent four-species dataset of primary white blood cells (n=26). Together, our findings support the argument that focusing on baseline states alone provides a biased view of gene regulatory evolution, and suggest that studying gene regulation in dynamic contexts can enrich our understanding of primate evolution.

Introgression between highly divergent species is generally selected against, yet some foreign alleles can break through species barriers when they provide a sufficient selective advantage, a process known as adaptive introgression. Here, we characterize the structural variant underlying adaptive introgression from Ciona robusta into C. intestinalis, two species separated by 14.5% net molecular divergence. Previous work identified a narrow peak of C. robusta ancestry on chromosome 5 that reached high frequency in several C. intestinalis populations despite otherwise restricted introgression. Using whole-genome Nanopore sequences from 15 C. intestinalis and three C. robusta individuals, together with short-read datasets including parent-offspring trios, we narrowed down the core introgressed segment from 16 to three genes and characterized copy-number variation affecting the cytochrome P450 gene CYP2U1c. The introgressed haplotype carries a tandem amplification of a 5,173-bp segment containing CYP2U1c, with 23 consecutive copies and conserved repeat boundaries. The nearly 120-kb array could not be fully resolved by long-read sequencing, but coverage consistently supported approximately 23 copies per introgressed haplotype. Despite its extreme size and repetitive structure, the allele showed Mendelian inheritance. CYP2U1c copy number was also polymorphic in C. robusta. Long reads fully resolved two structurally distinct haplotypes in one individual, carrying either a duplication encompassing adjacent CYP2U1b and CYP2U1c genes or five CYP2U1c copies. Their distinct breakpoints indicate independent origins. Our results reveal independently arising CYP2U1c amplifications in the donor species and show that an extreme high-copy-number allele was subsequently favoured following introgression into C. intestinalis.