Molecular Biology and Evolution

A fundamental unresolved question in molecular evolution is how novel genes arise from noncoding DNA and become fixed within stable gene repertoires. Here, we performed comparative genomic analyses across evolutionary timescales in insects using chromosome-scale genome assemblies of two cricket species, Teleogryllus occipitalis and Tarbinskiellus portentosus. Using conservative criteria, we identified 41 de novo gene candidates derived from intergenic regions in the Te. occipitalis lineage. These genes are simple and compact, exhibit hallmarks of evolutionarily young genes, and frequently contain fragments of transposable elements and simple sequence repeats. Across insects, such repetitive sequence fragments show positional homology but lack sequence conservation in older genes, suggesting that they serve as sequence material for gene emergence during early stages of gene evolution. In contrast, insertions after gene establishment are strongly constrained. We propose a model in which stages of gene evolution are characterized by shifts in selective pressure on the incorporation of sequence material.

Long runtime, high memory demands, and reliance on high-performance computing increasingly limit the evolutionary analysis of long phylogenomic datasets. We review a scalable framework based on phylogenomic subsampling and upsampling (PSU), in which many small subsamples of sites from a long concatenated sequence alignment are extended by upsampling prior to inference, and the resulting analyses are then aggregated to obtain stable evolutionary estimates. PSU exploits a useful distinction between the computational burden and the inferential power of statistical methods in molecular phylogenetics: computational cost is strongly influenced by the number of distinct site patterns in the concatenated alignment, whereas statistical power depends primarily on the amount of evolutionary information represented by sites and substitutions. By reducing the former while restoring the latter through upsampling, PSU can approximate many full-data analyses at substantially lower computational cost. Evidence from simulated and empirical datasets shows that PSU can accurately estimate bootstrap support values, select optimal substitution models, test evolutionary hypotheses, and infer branch lengths, divergence times, and associated uncertainty measures, while often reducing runtime and memory requirements by orders of magnitude. The same subsampling-upsampling-aggregation principle underlies all of these applications. PSU also provides distributions of inferred clade support across independent subsamples, enabling detection of concordant and conflicting phylogenetic signals that may remain hidden in conventional concatenated phylogenomic analyses. Adaptive procedures for selecting the subsample size, the number of subsamples, and the number of upsampling replicates make the framework practical across diverse datasets. We suggest that PSU is a general strategy for scalable phylogenomic inference across a broad range of statistical methods. By enabling rigorous analyses of genome-scale alignments on standard computing hardware, PSU expands access to computationally intensive evolutionary methods while reducing the environmental and infrastructural costs of big-data phylogenomics.

Whole-genome alignment (WGA) is widely used for genome-scale phylogenetic inference, and most scalable WGA pipelines rely on progressive alignment guided by a pre-specified tree. Among progressive whole-genome aligners, Progressive Cactus is a successful state-of-the-art method. However, analyses of real and simulated avian data indicate that guide-tree choice can influence downstream tree inference; star guide trees do not remove this effect and can exacerbate long-branch attraction artefacts. We have developed a consensus strategy based on the Progressive Cactus framework by generating a small set of alternative guide-tree alignments and retaining only homology relationships consistently recovered across all alignments. In simulation experiments, consensus alignments improve precision, bring inferred site-pattern frequency distributions closer to those of the true alignments, and recover more true splits than single guide-tree alignments. In a real landbird (Telluraves) dataset, we observe a strong bias towards single binary guide trees and long-branch attraction for less resolved trees. While the reconstructed tree still depends on the phylogenetic method and taxa sampling, our consensus alignment has no clear bias. We implemented a hierarchical consensus workflow that only locally resolves uncertainty in the guide tree. Therefore, the computational cost increases only moderately, for example by an estimated 68 percent for a recently published large-scale alignment of more than 300 modern birds (Neoaves) taxa.

Chemosensory systems play a central role in host detection, feeding behavior, and habitat selection in hematophagous insects. Here, we performed a comparative evolutionary analysis of chemosensory gene repertoires across 13 species of the Chagas disease vector genus Rhodnius. While gustatory receptors (GRs), ionotropic receptors (IRs), odorant-binding proteins (OBPs), and chemosensory proteins (CSPs) remained globally conserved, odorant receptors (ORs) displayed extensive lineage-specific expansions, tandem duplications, dynamic transcriptomic regulation, and recurrent signatures of positive selection. Major OR expansions were observed in Rhodnius robustus and Rhodnius colombiensis, suggesting increased sensory diversification in ecologically heterogeneous lineages. In contrast, conserved GR1 expression supports the maintenance of ancestral sugar-detection pathways despite hematophagy lifestyle. We further found no evidence of the canonical insect CO2-associated GRs, suggesting alternative molecular mechanisms for CO2 perception in Triatominae. Several receptors, including Orco, also displayed shifts in selective constraints between sylvatic and domiciliary species, consistent with sensory remodeling associated with adaptation to domestic habitats. Together, our results identify ORs as the most evolutionarily dynamic component of the Rhodnius chemosensory repertoire and highlight contrasting evolutionary trajectories among chemosensory gene families during ecological diversification and vector adaptation.

Comparative transcriptomic datasets are increasingly used to investigate the molecular basis of phenotypic diversification across species. However, finding genes that are differentially expressed (DE) between lineages remains challenging, for two main reasons. First, the random evolutionary drift can blur the signal left by lineage-specific shifts in mean expression, and induces phylogenetic correlations that, if ignored, can widely inflate the False Discovery Rate (FDR), i.e., the amount of spuriously detected genes. Second, DE analysis from RNA-Seq data involves multiple testing on many genes for a small number of individual measurements with high noise, and requires dedicated statistical tools. Traditional DE tools, such as limma, and classical Phylogenetic Comparative Methods (PCMs), such as the Expression Variance and Evolution (EVE) model, are both designed to tackle one of these two challenges alone, but both fail in the context of inter-species RNA-Seq data. In this work, we present phyloDE, a new tool for inter-species DE, that aims at taking the best from both approaches. On simulations based on a recently published four-species rodent dataset, we show that, contrary to other methods, phyloDE correctly controls the FDR in all settings, while keeping a reasonable power. When reanalyzing the empirical dataset, phyloDE discovers more DE genes that exhibit consistent changes in their cis-regulatory landscape compared to EVE in all the experimental settings. The method is implemented in R, with an interface inheriting from limma.

Given their rapid evolutionary dynamics, viruses offer a powerful system to investigate the mechanisms underlying host jumps. Here, we experimentally evolved endive necrotic mosaic virus (ENMV) in five plant hosts within the family Asteraceae: two putative ancestral hosts (Lactuca sativa and Tragopogon pratensis), and three alternative crop or weed species (Cichorium endivia, Zinnia elegans and Calendula arvensis). The resulting evolved viral populations, together with the ancestral strain, were then evaluated in a reciprocal cross-inoculation experiment across all five host species. ENMV exhibited clear adaptive responses in two hosts, Z. elegans and C. arvensis, with increased infection success and higher systemic viral accumulation compared to the ancestral virus. In contrast, no evidence of adaptation was detected in L. sativa, T. pratensis and C. endivia. Strikingly, strong cross-adaptation emerged between Z. elegans and C. arvensis: viral populations evolved in either host consistently outperformed those evolved in other hosts, as well as the ancestral strain, when infecting the reciprocal host. Sequencing of the VPg cistron in adapted populations revealed multiple nonsynonymous mutations, several of which arose independently across evolutionary lineages and in both Z. elegans and C. arvensis selection regimes. Functional assays using an infectious ENMV cDNA clone demonstrated that seven of these substitutions, individually or in combination, significantly increased the infection rate in both Z. elegans and C. arvensis. Notably, several of these substitutions also enhanced infectivity across four additional Asteraceae species among the eleven tested, without a clear relationship to host phylogenetic distance. Remarkably, all identified substitutions map to amino acid positions or adjacent residues in VPg previously implicated in the breakdown of recessive resistance genes against potyviruses in both crop and model plant systems. Together, these results suggest that adaptation to host resistance and host range expansion in potyviruses may rely, at least in part, on shared molecular pathways.

Whole-genome duplication (WGD) is hypothesized to spur evolutionary diversification by producing genome-wide duplicate gene sets (Ohnologs) that are initially functionally redundant but can diverge markedly as the effects of relaxed selection accumulate over time. However, the underlying mechanisms remain unclear, in part because genomic studies often reconstruct Ohnolog evolution over millions of years, during which subsequent mutations can obscure deep-time signals. Investigating the relationship between Ohnolog evolution and diversification on a contemporary timescale offers clearer insights. We explore this relationship in Lake Charr (Salvelinus namaycush), where ~10% of genes are retained highly conserved polyploid duplicates following the Salmonid-Specific Fourth Round WGD. Using 31 chromosome-level assemblies of Lake Charr from morphologically and ecologically diverse populations, joined into a pangenome graph, we characterized 189,555 structural variants (SVs) that were significantly less likely to affect genes retained as sequence-conserved Ohnolog pairs, nuancing the hypothesis that gene redundancy, relaxed selection, and functional diversification are intertwined. However, we found that SVs affecting such conserved Ohnologs may be potent drivers of adaptive evolution. Notably, we identified a putative 938-Kb interchromosomal translocation containing 25 genes with highly conserved Ohnologs in a paralogous (but untranslocated) genomic block. This putative translocation appears to have facilitated Ohnolog divergence in ankrd11 and hp, genes putatively linked to craniofacial and lipid metabolic diversity in sympatric Lake Superior morphs. This research reveals that conserved Ohnologs previously presumed to be redundant remain a reservoir for adaptive change.

Rare genomic changes have long been sought by phylogeneticists for their potential to resolve obdurate nodes in the tree of life. Recently, patterns of macrosynteny have been proffered as a breakthrough for challenging relationships within invertebrates. One taxon that stands to benefit from the application of this approach is Chelicerata (the sister group to the rest of Arthropoda), whose radiation has long defied resolution, despite intensive investigations using morphological characters, molecular sequence data, and a combination thereof. Challenges to the resolution of chelicerate phylogeny include an ancient rapid radiation, the incidence of several fast-evolving lineages prone to long-branch attraction artifacts, and extinction of multiple ordinal level lineages that cannot be sampled for breaking long branches. At present, only a subset of nodes has been stably resolved. To break this impasse, we brought to bear multiple classes of phylogenetically informative rare genomic changes, including the sequencing of the first genomes for Ricinulei and Palpigradi. Here, we show that an ancient, shared whole genome duplication event is restricted to Arachnopulmonata (the most recent common ancestor of spiders and scorpions), disfavoring traditional placements of either Ricinulei or Palpigradi as close relatives of tetrapulmonates. Intriguingly, investigation of fusion-with-mixing events identified equal support for mutually exclusive placements for Acariformes, the least stable of the arachnid orders. Our results suggest that fusion-with-mixing, far from being a silver bullet, likely exhibits the same emergent property as all character systems, in that it is prone to homoplasy and conflicting signal stemming from ancient rapid radiations.

Gene regulatory divergence has emerged as a key feature in speciation, influencing gene expression differences that accumulate between diverging populations. Transcriptional regulation, mediated by cis- and trans-acting factors, modulates diverse developmental processes and is responsible for distinct species-specific gene expression profiles. Within interspecies hybrid individuals, negative interactions between divergent cis- and trans-acting factors can lead to gene misregulation and hybrid dysfunction at the organismal level. Such gene regulatory mismatch might disproportionately impact sex-biased and tissue-biased gene regulatory networks due to their unique selective pressures. To address these issues, we investigated the role of regulatory divergence in asymmetric hybrid incompatibility between sister species of Caenorhabditis nematodes (C. remanei, C. latens) by analyzing gene expression of reciprocal hybrids for each sex and key tissue types. Despite severe hybrid male sterility, hybrid males showed less misexpression of sex-biased genes than hybrid females, suggesting that the organismal phenotypic outputs of male-biased gene regulatory networks are more vulnerable to disruption than female-biased genetic networks. Additionally, we found more genes associated with cis- than trans-regulatory divergence, supporting the notion of a disproportionate role for cis-regulatory divergence between species. Moreover, we document extensive cis-trans compensatory X-linked regulatory divergence specifically from male transcriptomes, indicating distinct molecular evolutionary outcomes of stabilizing selection on regulatory controls in males and females. These insights derived from asymmetric hybrid misexpression expand our understanding of the evolution of sex-biased gene regulation in the face of stabilizing selection and identify candidate genes contributing to Caenorhabditis post-zygotic reproductive isolation.

Although DNA Large Language Models (DNA-LLMs) offer a path to decoding genetic complexity, our ability to evaluate these models is constrained by our incomplete understanding of the very same genetic syntax and functional logic that these models are trained to learn. In this study we use single nucleotide substitutions that have or have not been observed in living organisms, to evaluate how the DNA-LLM Evo 2 interprets gene sequences from two plant model organisms, Arabidopsis thaliana and Oryza sativa japonica. Using perplexity as a measure of the model's confidence, we observe that alleles containing simulated substitutions are perceived, on average, as less likely than those observed in vivo. Although the size of the effect is modest, the effect is statistically significant and robust, suggesting that Evo 2 is aligned with our current understanding of evolutionary selective constraints. This approach is designed to be model-agnostic and species-agnostic and could serve as a generic framework for evaluating the performance of DNA-LLMs.

Novel antagonistic interactions between species are expected to drive especially rapid coevolution. However, little is known about the genomic basis of such coevolution in nature because novel inter-specific interactions are rarely observed. Here, we study two species that recently came into first contact in Hawaii, the parasitoid fly Ormia ochracea and its cricket host Teleogryllus oceanicus. The fly locates crickets acoustically using their song, and parasitism usually results in host death. In response, protective male-silencing mutations have rapidly spread through cricket populations over the last ~25 years, imposing novel selective pressure on flies. By integrating population genomic analyses of 358 re-sequenced flies with field surveys of selection imposed by host adaptations, we discover genomic signatures of recent selective sweeps driven by host adaptations, indicative of escalating arms-race dynamics. This evolutionary response is occurring despite severely depleted genetic variation after bottlenecks in Hawaiian fly populations. Comparative analyses suggest that the genomic substrate of modern-day, rapid counter-adaptation in O. ochracea has been under positive selection on intermediate and long-term timescales across parasitoid flies. Our findings thus support predictions of influential arms race coevolution models and illustrate the current and ancient genomic bases of counteradaptation in nature.

Codon usage bias is a central record of mutation, selection, drift, and translational constraints, but it is usually treated separately from generalized Chargaff symmetry, the tendency for words and their reverse complements to occur at similar frequencies in long DNA sequences. Here we ask whether codon usage contains a measurable reverse-complement component, and whether departures from that component can be quantified. We first avoided imposing reverse-complement symmetry. Instead, we exhaustively evaluated all nontrivial symbolic involutions (f(f(x)) = x) of the 64 codons, constructed from self-inverse nucleotide maps and self-inverse codon-position permutations. Across taxa, the reverse-complement transformation was the optimum, giving the highest median correlation between codon frequencies and transformed codon frequencies. Random and amino-acid-preserving reference models showed that the signal is not a generic property of codon profiles and is only partly explained by protein composition. Additional controls preserving amino-acid composition and matching GC3 in expectation showed that the observed reverse-complement correlation remains higher than expected from these constraints alone, and genus-level aggregation confirmed that the optimum is not driven by overrepresented genera. The symmetry breaks in a biologically ordered manner: the third, most degenerate codon position remains closest to the reverse-complement baseline, whereas the first position departs most strongly and the second is intermediate and lineage dependent. Taxonomic comparisons reveal broad and fine-scale heterogeneity in the degree of codon-level symmetry preservation. Together, these results show that codon usage can be quantified as a combination of reverse-complement preservation and position-, lineage-, and function-dependent departures from the GCT-associated compositional baseline, linking sequence-level symmetry, evolutionary mechanisms, and codon-level organization within a single measurable framework.

Mesodinium rubrum, a marine microbial eukaryote associated with some of the largest red tides on Earth, has the remarkable ability to commandeer the plastids, mitochondria and nuclei from the alga Teleaulax amphioxeia for photosynthesis. Here we report analyses of assemblies of M. rubrums two nuclear genomes. Unexpectedly, M. rubrum appears to have completely lost its spliceosomal introns, most spliceosomal molecules, and the key genes for an intron splicing-associated process, Nonsense-mediated mRNA Decay (NMD). In contrast, non-spliceosomal tRNA introns have been retained, as have thousands of intron analogs spliced out of DNA during ciliate somatic genome development (internal eliminated sequences - IESs). Intron-containing genes, especially from intron-rich species like T. amphioxeia, would likely be defunct if horizontally transferred to a host without a spliceosome like M. rubrum, and thus we propose that introns can be a roadblock to progressive endosymbiotic genomic integration.

Reconstructing patterns of evolution requires understanding the interrelationships of species, yet evolutionary relationships that defy resolution and calibration in time are commonplace across the Tree of Life. Here, we investigate the dynamics of temporal and topological uncertainty by generating a phylogeny of jawed vertebrates using 1105 exonic loci sampled for 540 species spanning all major orders and most families of gnathostomes. Across loci and DNA sequence sites, we observe rapid reductions in statistical support for the monophyly of jawed vertebrate clades that originated around the Cretaceous-Paleogene mass extinction. Phylogenetic signal was scrambled to different degrees during rapid successive divergences in multiple unrelated jawed vertebrate lineages that radiated in this interval, including birds, snakes, placental mammals, and acanthomorph fishes. In addition to showing that particular events have modified phylogenetic signal across the same loci in distantly related vertebrate clades, we also demonstrate how rates of genomic evolution affect our ability to infer the timescale of vertebrate evolution. By testing how the inclusion of lineages of ray-finned fishes with very fast and slow rates of molecular evolution changes inferences of the vertebrate evolutionary timescale, we show that the deepest divergences in ray-finned fishes may be impossible to accurately infer using sequence data and calibrations from a limited fossil record. These results hint at the macroevolutionary realities underlying topological and divergence time uncertainty across evolutionary trees.

Interactions among individuals in structured populations can alter fitness effects of mutations and reshape evolutionary processes. In many systems, including bacteria, yeast, and viruses, such interactions often result in public goods: gene products that are costly to produce yet exploitable by others. During viral coinfection of the same cell, gene products from one genome may complement deleterious mutations in another, allowing defective genomes to persist. Yet it remains difficult to infer which proteins are shareable from population sequencing data, because mutation, selection, drift, and complementation are intertwined. Here, we developed a quantitative framework to infer protein-specific public goods in the RNA bacteriophage MS2, which encodes only four proteins. We analyzed experimental evolution data generated under two multiplicity-of-infection (MOI) regimes: low MOI, where coinfection is rare, and high MOI, where coinfection is common. We first compared empirical mutation patterns between regimes and then applied a Wright-Fisher model combined with simulation-based Bayesian inference using neural posterior estimation. In a two-stage strategy, gene-specific fitness effects were inferred from low-MOI data and subsequently used to estimate protein sharing under high-MOI conditions. Across two statistical inference frameworks, lysis emerged as the strongest public-good candidate, replicase and coat showed an intermediate signal, and maturation showed the weakest evidence for sharing. Together, our results show that viral proteins differ markedly in their propensity to act as public goods. More broadly, they illustrate how coinfection can generate density-dependent selection, a general feature of social evolution that may shape evolutionary dynamics.

Germline restricted chromosomes (GRCs) are an evolutionary mystery. While they seem to be important and perhaps indispensable in species they occur, they also show surprising variability in copy number and size. We are just beginning to understand the evolutionary significance of these chromosomes. One factor that impedes our understanding is that at present, few clades with these chromosomes have been studied in depth. We genomically investigate the GRCs in dark-winged fungus gnats (Sciaridae), a cosmopolitan and species-rich taxa which have harboured GRCs for at least 58 million years. Sciaridae species carry from 1-4 large GRCs which evolved through introgression from the Cecidomyiidae lineage. We produce the first male germline chromosome-scale genome assemblies for the sciarids Bradysia coprophila, B. impatiens, and Lycoriella ingenua. Comparing the GRCs both between and within species, we find that these chromosomes evolve in an extraordinarily dynamic fashion, with very little conservation of synteny or gene content. Puzzlingly, despite this variability, GRCs are gene rich and their genes are evolving under strong purifying selection. Additionally, we uncover evidence that somatic elimination of GRCs potentially occurs via a satellite mediated process. Our study sheds light on the mechanisms of meiosis, tissue restriction, chromosome elimination, and gives us a better appreciation for the diversity of genetic systems that not only persist, but thrive in nature.

Many successful lineages, including flowering plants and vertebrates, owe some of their evolutionary prosperity to whole genome duplications (WGD). However, in the immediate aftermath of a WGD, the new polyploid species that is formed often experiences multivalent pairings during meiosis, which can produce inviable gametes. To mitigate the potential harm caused by such pairings, most lineages eventually undergo "diploidization" to restore typical bivalent pairing. A key component of this process is the loss of duplicated genes. While diploidization was once thought to be rapid, recent analyses of polyploidies suggest the process may be more drawn out, with multivalent pairing persisting long after the initial WGD event. Here, we assess evidence for "late" diploidization after three different polyploidies: the teleost genome duplication (TGD), nested polyploidies in Paramecium lineages, and the ancient WGD in bakers yeast. Using our tool POInT (the Polyploidy Orthology Inference Tool), we model the resolution of these events. By analyzing discordance between expected species trees and observed gene trees, we argue that late diploidization was a likely feature in the resolution of all three polyploidies.

Long-term studies of isolated animal populations have greatly improved the understanding of various evolutionary processes. However, potentially elevated inbreeding in those compared to wild populations is a common concern. Conventionally, inbreeding has been investigated using reconstructed pedigrees, but nowadays it can be done directly at the genomic level. Here, we utilize genomic data from an intensively studied isolated rhesus macaque (Macaca mulatta) population on the small island Cayo Santiago (Puerto Rico), which was founded in 1938 with wild animals from India. We quantified inbreeding levels by inferring runs of homozygosity (ROH), i.e., long identical haplotypes inherited from both parents. We identified ROH in 97 ~5x-coverage genomes from Cayo Santiago and, for comparison, in 79 rhesus macaque genomes from five wild populations from China. Notably, this conventionally considered low-coverage data proved sufficient to infer ROHs >4 centimorgans long after imputing the genomes using a reference panel. Our results revealed that the ROH-derived effective population size on Cayo Santiago, 420 individuals, falls within the ranges we inferred in wild populations. Moreover, a general scarcity of individuals with long ROH in both the Cayo and wild populations indicates very few cases of close-kin breeding, suggesting that mechanisms to avoid close-kin breeding operate in rhesus macaques, both in wild and isolated populations. Taken together, our results suggest that Cayo Santiago remains a representative study population.

Aedes aegypti is an exceptionally effective global vector of human disease because of its strong specialization on human hosts and habitats. However, in its native range in Africa, many populations never specialized on humans and retained an ancestral generalist ecology. Now, in the rapidly growing cities of Kumasi, Ghana and Ouagadougou, Burkina Faso, we directly document genomic and behavioral evidence of a sudden shift towards greater specialization on humans over just five years. These changes are likely to enhance the ability of these urban mosquito populations to serve as effective vectors of human disease and may have played a role in unprecedented recent outbreaks of dengue fever in West African cities.

Demographic inference using the Site Frequency Spectrum (SFS) is often constrained by the number and complexity of models tested. Here we present a coalescent model generator called CoalMiner for use with fastsimcoal2. CoalMiner utilizes a decision tree framework to generate biologically plausible models, with user input dictating the number and ranges of demographic parameters and histories, which can then be plugged into the fastsimcoal2 pipeline. Using extensive simulations and empirical data, we show that CoalMiner is an effective helper tool to explore demographic model space. CoalMiner is written in Python and is freely available on GitHub: https://github.com/raywray/coalminer with numerous vignettes and tutorials.