Asexual lineages are thought to experience elevated extinction rates compared with sexual species, yet direct evidence for the underlying genetic causes remains scarce. Muller's ratchet predicts that the absence of recombination in asexual organisms facilitates the accumulation of deleterious mutations, thereby reducing long-term fitness. Here, we test this hypothesis in the hybrid-origin, parthenogenetic whiptail lizard Aspidoscelis tesselatus by integrating short-read RNAseq and long-read IsoSeq data from both the asexual lineage and its parental sexual species. We reconstructed phased transcripts for A. tesselatus to quantify mutation accumulation relative to the parental sexual species. Comparative analyses revealed elevated ω ratios in both parental genomic complements (subgenomes) of the parthenogenetic lineage, consistent with accelerated accumulation of nonsynonymous mutations. Structural variant analyses identified multiple indels in expressed transcripts predicted to disrupt protein domains. Functional annotation indicated that genes affected by both single-nucleotide variants and indels were enriched for roles in chromatin organization, apoptosis regulation, and transcriptional control. While both parental subgenomes showed similar evolutionary patterns, the maternal complement exhibited more structural and missense mutations than the paternal complement. Together, these results provide evidence that mutations accumulate in asexual A. tesselatus in genes involved in core cellular functions, supporting theoretical predictions that Muller's ratchet contributes to mutation accumulation in asexual lineages.
Estrogen receptor-positive breast cancer (ER+ BC) is one of the most prevalent cancers, but the evolutionary processes shaping genetic variation in ER+ BC risk are poorly understood. Both evolutionary life-history theory and evidence from studies of individual ER+ BC risk variants suggest that increased genetic ER+ BC risk is associated with faster maturation, earlier reproduction, and/or increased reproductive success (i.e., there is a trade-off), but it is unclear how well this pattern is replicated when considering the polygenic architecture of these traits after controlling for potential biases. Here, we estimate genome-wide genetic correlations between ER+ BC risk and three reproductive traits (age at menarche, age at first birth, and the number of children) using genomic restricted maximum-likelihood analyses on Lifelines biobank data and linkage disequilibrium score regressions on population and family-based genome-wide association study data. Regardless of the data or method used, genetic correlations were low and not statistically significant. Further analyses decomposing genome-wide genetic variance into local regions detected only three loci exhibiting significant pleiotropy between ER+ BC risk and age at menarche, suggesting little shared genetic architecture between ER+ BC risk and reproductive traits. Thus, the role of life-history trade-offs in shaping ER+ BC risk in European populations appears, at most, small, and the evolutionary processes giving rise to this life-threatening disease remain unclear. Future studies could examine the impact of evolutionary mismatches in shaping ER+ BC risk, where conducting longitudinal studies on populations transitioning to reproductive patterns observed in contemporary European populations would be most useful.
Natural host populations are age-structured, and developmental stages differ in susceptibility and within-host pathogen dynamics, potentially imposing distinct selective pressures on viruses. However, the evolutionary consequences of host age structure remain poorly understood. We experimentally evolved turnip mosaic potyvirus for five passages in Arabidopsis thaliana populations spanning seven demographic regimes, from juvenile- to mature-dominated cohorts. We quantified disease progression, symptom severity, and viral load, cross-inoculated evolved lineages across host stages to construct infection matrices, and performed whole-population sequencing at passages 1 and 5. Disease traits changed markedly with passage, demography, and their interaction. Disease progression evolved faster in older populations, whereas symptom severity was independent of median age, indicating demographic reweighting of virulence components. Viral load increased across passages and positively correlated with severity, linking within-host fitness to symptoms. Cross-inoculation assays revealed a modular infection network: juvenile-evolved lineages specialized on juvenile hosts, whereas lineages from intermediate and older populations were more generalist. Genomically, we detected both parallel and demography-specific adaptations, including recurrent changes in the viral protein VPg (involved in translation, replication and host interactions) as well as synonymous variants showing consistent or opposing selection across host population stage structures. Overall, host age structure emerges as a major ecological driver of virulence evolution, shaping trade-offs between disease progression and severity and determining specialization versus generalism. These results integrate phenotypic and genomic responses and suggest that manipulating crop age structure could steer virus evolution toward less damaging outcomes.
Conserved water molecules (CWMs) are tightly bound solvent molecules that occupy well-defined, recurrent positions in protein structures. Although they are known to influence protein stability, function, and ligand binding, their role in shaping the effects of human missense variants remains largely unexplored. Here, we demonstrate that CWMs are a previously underappreciated determinant of missense variant pathogenicity. By predicting ligand-binding and CWM sites across human PDB structures and mapping missense variants to these sites and the remaining protein surface, we found that pathogenic variants were significantly enriched at CWM sites, whether overlapping or outside other ligand-binding regions. This enrichment exceeded that observed for binding sites as a whole, indicating a broader role for water-mediated interactions in modulating variant effects. To explore a mechanistic basis for this association, we performed molecular dynamics simulations of human lysosomal acid glucosylceramidase (GCase), encoded by GBA1 and implicated in Gaucher disease and Parkinson's disease risk. Selective destabilization of a CWM site in wild-type GCase produced structural and dynamical changes resembling those observed in the pathogenic L444P variant, whereas stabilization of this site in L444P shifted several measures toward wild-type behavior. These results suggest that disruption of a single CWM can contribute to long-range structural remodeling observed in a disease-associated variant. Together, our findings identify CWMs as a novel structural constraint shaping the distribution and effects of pathogenic missense variants. Incorporating water-mediated interactions into structural models provides a generalizable framework for interpreting human genetic variation and its contribution to disease.
Saliva forms the first biochemical interface with the environment, mediating dietary exposure and oral defense. Proline-rich proteins (PRPs) in saliva protect plant-eating mammals from the harmful effects of consuming tannins. Yet the evolution of the genes encoding these proteins remains poorly resolved. Here, integrating long-read genome assemblies with comparative genomics, phylogenetics, transcriptomics, and dietary data, we investigate the salivary PRP gene family across 27 primate species to test whether dietary ecology has shaped the evolution of this locus. We find that the PRP locus is dynamic, shaped by recurrent lineage-specific gene duplications and inversions. Despite pervasive structural variation, PRP genes consistently rank among the top five most highly expressed transcripts in non-human primate parotid glands, indicating constraint on maintaining high expression in saliva. Humans retain abundant PRP expression, but at reduced levels relative to other primates, potentially reflecting a lineage-specific regulatory shift. We further identify widespread exonic tandem repeats that provide a flexible mechanism for modulating protein composition. Notably, colobine PRH genes independently evolved trinucleotide repeats encoding histidine-rich peptides, coinciding with folivory and suggesting a lineage-specific adaptation. Together, our results position PRPs as a model for understanding how large-scale structural rearrangements and fine-scale repeat expansions jointly shape the diversity and evolution of proteins in saliva.
Transcriptional adaptation (TA) is a regulatory process in which loss or disruption of gene function caused by protein-truncating variants (PTVs) triggers compensatory changes in expression of the healthy allele or in expression of related genes. Nonsense-mediated mRNA decay (NMD), a conserved RNA surveillance pathway that degrades transcripts containing premature termination codons, is required to initiate this process. In natural yeast populations, PTV mutations occur relatively frequently, raising the question of whether and how their effects are mitigated. In this study, we investigated TA and NMD among PTVs occurring in natural yeast populations. We observed a strong reduction in the abundance of PTV-containing transcripts, suggesting efficient recognition and degradation of transcripts containing premature stop codons. However, despite evidence of this mRNA surveillance activity, we did not detect a clear signature of transcriptional adaptation. While compensation may still occur in specific contexts, particularly for dosage-sensitive genes, it does not appear to represent a general response to PTVs in yeast. The transcriptional deregulation caused by PTVs in natural isolates may be causing too little harm to favor the evolution or maintenance of complex mechanisms required for adequate compensation. The ability to resist specific transcriptomic ruptures would thus rely mostly on the general robustness of genetic networks. Overall, our findings suggest that TA is not a universal response to loss-of-function mutations in yeast.
Mitogenomic datasets are central to molecular evolution and phylogenetics, yet preparatory workflows remain labor-intensive and prone to errors introduced during manual data preparation. EZmito2 is a re-implementation of the widely used EZmito pipeline that offers a fully reproducible and user-accessible solution for mitogenomic dataset curation and result visualization. It is accessible via a public web server for rapid analyses and through local installation, enabling reproducible workflows on personal computers or computational clusters. The pipeline consolidates the core modules-EZpipe, EZskew, and EZcodon-and extends functionality through newly developed tools for genome visualization (EZcircular, EZmap), chimeric region detection (EZmix), gene extraction from NCBI-deposited genomes (EZsplit), structural annotation of transmembrane domains in mitochondrial protein-coding genes (EZtrampo), and population genetic studies (EZdist, EZpcoa, EZpopstat). All tools accept standard input formats and generate ready-to-publish outputs. By providing a user-friendly platform for mitogenomic exploration and quality control, EZmito2 facilitates reproducible analyses for evolutionary and molecular research communities.
The fossil record is incomplete, as evidenced by the pervasive presence of ghost lineages throughout the Tree of Life. For example, across placental mammals, at least 720 Myr of basal lineages are ghost lineages, that is, lineages that have left no fossil evidence of their past history. In contrast, some studies have suggested that the fossil record is a faithful temporal archive of evolutionary history and thus the times of diversification of clades must be close to the ages of their oldest fossils. Such literalist interpretations have been contradicted by analysis of molecular datasets which, in many cases, indicate that groups including placental mammals and animals may have originated at times substantially older than their fossil records. Some of those studies have further argued that, in the case of animals and placental mammals, molecular clocks are uninformative, suffer from characteristic pathologies, and thus cannot distinguish between recent and ancient hypotheses of diversification. Here, we reexamine these two cases and show, using Bayesian model selection theory, that the explosive diversification models previously proposed for animals and placental mammals have a posterior probability of ∼0. We show the characteristic pathologies purportedly discovered do not exist, highlight errors in previous analyses, and provide advice on best practice for molecular-clock dating analysis.
Comparative genomics provides a powerful framework to uncover the molecular and evolutionary mechanisms that shape genetic diversity, revealing how shared or lineage-specific processes influence their evolutionary trajectories. The nematode Caenorhabditis briggsae is distributed world-wide and is a comparative model to Caenorhabditis elegans in the biology of development, cellular mechanisms, neurobiology, complex trait mappings, and evolution. Following massive collection efforts by the nematode research community, we present the isolation of over 1,900 wild strains and analyses of genome sequences that catalog over six million single-nucleotide and insertion-deletion variants. These resources provide a powerful means to interrogate the causal genetic bases of phenotypic variation. Additionally, we describe C. briggsae population structure and discover new, genetically distinct groups within this primarily self-fertilizing species, including groups of highly related strains sampled across entire continents. We leveraged expansive genetic variation to decipher the effects of linkage and selection on the distribution of genetic diversity across the genome and across geographic regions. Within the species, we find genomic regions with extremely high levels of genetic variation similar to hyper-divergent regions found in C. elegans and other species. These regions harbor new genes and variation enriched for environmental sensing and pathogen responses. Based on comparisons to the outbreeding sister species Caenorhabditis nigoni, we conclude that long-term balancing selection has maintained substantial functional variation, likely associated with ecological variation, within C. briggsae since its divergence from an outbreeding ancestor. Overall, this massive strain resource enables future comparative genetics studies, including genome-wide association study contrasts between Caenorhabditis species.
We investigate the predictability of evolution in terms of the phylogenetic placement of new lineages. This leads us to develop a class of coalescent models that relax neutrality by allowing the rate of coalescence to vary as a continuous heritable trait. In this setting, each lineage has a relative propensity to coalesce, with coalescent odds ratios defined from the product of pairwise propensities. Estimated coalescent odds provide a statistic that captures variation in lineage growth and are informative about the strength of natural selection acting on individual lineages. A number of practical statistical methods are then developed: techniques to adjust for biased and nonuniform sampling; procedures to automatically calibrate hyperparameters governing the evolution of coalescent propensity; and methods for clustering phylogenies into sets that delineate clades according to coalescent propensity. Simulations show sensitivity of these methods to detecting small selective effects acting on rare variants and strong robustness to imbalanced sampling. We demonstrate these methods using two datasets reflecting microbial populations evolving under strong selection. First, we examine a large set of Neisseria gonorrhoeae genomes and show that lineages with high coalescent odds feature a unique antibiotic resistance pattern, which presaged its subsequent expansion. We then reanalyze SARS-CoV-2 data in combination with independent estimates of reproduction numbers corresponding to major variants of concern 2020-2023. This indicates that coalescent odds can function as an excellent tree-based proxy for relative fitness of major SARS-CoV-2 lineages.
Standard probabilistic models of coding sequence evolution effectively identify where and when selection acts but remain agnostic to the mechanistic realization of these forces. We introduce PRIME (PRoperty Informed Models of Evolution), a framework of codon-level maximum likelihood methods-including global (G-PRIME), episodic (E-PRIME), and site-specific (S-PRIME) implementations-that explicitly model amino acid exchangeability as a function of physicochemical properties. By parameterizing attributes such as molecular volume, hydropathy, and secondary structure propensities, PRIME aims to resolve the biophysical basis of selective constraint across both the sequence and the phylogeny. At the site level, S-PRIME leverages an explicit biophysical taxonomy to categorize residues as conserved, neutral, or changing for specific properties, resolving selective signals that are missed by traditional rate-based metrics. Our analysis of a benchmark of 24 diverse datasets and a genome-wide screen of 18,944 mammalian genes demonstrates that consideration of biophysical realism can yield substantial improvements in model fit, acting synergistically with rate variation to explain complex evolutionary patterns. We find that physicochemical constraints at individual sites can be reliably detected in datasets with sufficient information redundancy (substitutions per unique amino acid; AUC=0.91), with sensitivity exceeding 90% in data-rich alignments. E-PRIME reveals a distinct hierarchy in biophysical constraints: while core packing and beta-sheet scaffolds are rigidly conserved, alpha-helix propensity and surface electrostatics serve as the primary substrates for adaptive tuning. Furthermore, PRIME importance weights align with aspects of the primary semantic axes of deep learning representations (ESM-2) and capture key features of experimental fitness landscapes. By transforming abstract evolutionary rates into interpretable biophysical rules, PRIME provides a useful framework for characterizing the mechanistic drivers of protein diversity.
Uncovering the regulatory architectures that underlie complex phenotypes can provide insight into both the mechanisms and evolution of unique adaptations. In bears, thousands of genes are differentially expressed in a tissue-specific manner during hibernation, many of which are involved in major vertebrate metabolic signaling pathways. However, the precise regulatory mechanisms driving these gene expression changes, and the extent of their conservation in non-hibernating mammals, remain poorly understood. Using capped-small RNA-sequencing from brown bear adipose tissue, we identify putative enhancers that exhibit dynamic shifts in regulatory activity during hibernation. The majority of these enhancers share sequence homology with known human enhancers, yet many appear to target distinct genes, suggesting a role of regulatory co-option in the evolution of hibernation. Using these newly identified enhancers, we identify transcription factors putatively underlying hibernation gene expression, expanding our mechanistic understanding of hibernation physiology. Additionally, we find evidence for selection on cis-regulatory sequences associated with physiological adaptation across bears. Collectively, this study provides new perspectives on the mechanisms and evolution of mammalian hibernation, and the roles of regulatory sequences in the evolution of complex physiological adaptations.
Phenotypic plasticity allows a single genotype to maintain its fitness across different environments. This facilitates colonization of new niches but can be further refined or even lost as lineages diversify. The toxic Heliconiini butterflies have biochemical plasticity: they either sequester their cyanogenic glucosides (CGs) from their larval host plant or biosynthesize them when compounds for sequestration are not available. Here, we trace the evolution of CG biosynthesis in Heliconiini butterflies, a fundamental component of this biochemical plasticity. We first reconstructed the evolutionary history of biochemical plasticity in Heliconiini using chemical data from over 700 individuals, demonstrating that plasticity was ancestral in the tribe but subsequently lost in a few clades, such as the Sapho clade specialized in CG sequestration. In lepidopterans, CG biosynthesis has previously been characterized in the moth Zygaena filipendulae, as the genes CYP405A2, CYP332A3, and UGT33A1. Thus, we CRISPR-edited CYP405 in Heliconius erato and confirmed that CYP405-knockout caterpillars do not biosynthesize CGs. We identified the CYP405As and CYP332As in other lepidopterans and found that both genes were independently co-opted into CG biosynthesis in the Heliconiinae butterflies and Zygaena moths. While most lepidopterans have a CYP332A, CYP405A is mostly restricted to butterflies and has been duplicated in all Heliconius species. Although several CYP405A copies were found in the Sapho clade, most of them lack structurally important P450 domains, which explains the loss of biochemical plasticity via specialization in CG sequestration. This is one of the few examples of plasticity-first evolution in which the genetic mechanisms associated with its refinement are known.
Following the evolution of internal fertilization, the female reproductive tract became the site of two major reproductive interactions, the insemination response and the long-term postmating response. However, our understanding of these two responses is hampered by the fact they coincide in most animals. Traumatic insemination in the common bedbug (Cimex lectularius) presents a unique opportunity to disentangle these effects because females have evolved a novel organ, the mesospermalege, which is the site of insemination, while sperm do not arrive in the reproductive tract until several hours after mating. Here, we show that the mesospermalege exhibits a gene expression profile consistent with functions of the insemination response, including immune responses, that are normally found in the lower reproductive tract in other insect species. We then show that the postmating response in the lower female reproductive tract of bedbugs is delayed, coinciding with the movement of sperm through the female, suggesting that the postmating response has evolved in response to sperm receipt independent of intromission and insemination. Finally, we show male seminal fluid genes are expressed in the female, previously only demonstrated in Drosophila. Seminal fluid genes are more highly expressed in the mesospermalege than the reproductive tract, suggesting co-expression has evolved as part of the insemination response. Our results provide insights into the evolution of novel organs, reproductive traits, and female postmating gene expression in a global pest with an unusual reproductive biology.
Differences in ploidy impact fitness, shaping adaptation and evolutionary trajectories. Although polyploidy has been documented in corals, the mechanisms that give rise to polyploidy and the phenotypic consequences of this variation are unknown. We established a model population of 326 Pocillopora acuta colonies in Kāne'ohe Bay, Hawai'i, and used RAD sequencing of these samples and historical collections to define clonal lineages, ploidy state, and mechanistic origin of polyploidy in each colony. We also measured growth, morphometrics, thermal tolerance, and fecundity in common-gardened corals. Here, we show that triploidy in P. acuta likely originated via allopolyploidy via hybridization between P. acuta and its sister species, P. damicornis. Triploids were present in this population before and after the 2014/2015 bleaching events, becoming the dominant ploidy on contemporary reefs (>60% of genotypes). These bleaching events did not select for triploid genotypes; however, relative triploid abundance may have increased in part due to higher growth rates compared to diploids. Both ploidies released larvae on the same lunar cycle and there was no difference in photosynthetic efficiency during heat stress, but triploid colonies experienced greater mortality at lower levels of stress, suggesting a host-derived fitness consequence influenced by ploidy state. This work highlights a cryptic source of variance in coral fitness, with important implications for understanding their ecology and evolution.
The surface layer of the skin and hard skin appendages of land-dwelling vertebrates depend on epithelial differentiation-associated proteins, which increase the mechanical resilience of cells upon cornification. These proteins, exemplified by keratin-associated proteins (KRTAPs) in mammals and epidermal differentiation complex (EDC) proteins in all tetrapods, have low amino acid sequence complexity and interact with other proteins to form stable supramolecular structures. No KRTAPs and only few EDC proteins exist in amphibians, suggesting an alternative mechanism of epithelial cornification. Here, we used comparative genomics and proteomics to identify a previously uncharacterized group of epidermal differentiation-associated proteins that are specific for amphibians. These proteins are encoded by genes of the amphibian epidermal differentiation cluster (AEDC), which has evolved by tandem duplications and sequence modifications of an ancestral gene at a locus syntenic with that of adipogenesis regulatory factor in other vertebrates. AEDC genes are specifically expressed in the epithelium of the skin and its appendages, such as the cornified spines within nuptial pads of frogs. Internal sequence duplications within AEDC proteins have led to amino acid sequence repeats similar to those of EDC proteins and KRTAPs. We conclude that epidermal differentiation-associated proteins have originated multiple times and evolved similar sequence features in amphibians and other clades of vertebrates.
The evolution of visual systems has compelled numerous investigations of developmental processes underlying eye patterning across Bilateria. It is well-established that homologs of the transcription factor Pax6 play a highly conserved role in eye fate specification and are at the top of the retinal determination gene network (RDGN) hierarchy. Curiously, Pax6 homologs do not appear to maintain this function in well-studied chelicerate models. It was recently proposed that the gene Pax2 may have subsumed the role of Pax6 in eye fate specification in chelicerates. However, no functional data are available for any chelicerate Pax homologs. We examined the incidence of Pax family genes across chelicerate phylogeny, and interrogated the expression and function of Pax2 and Pax6 homologs in the daddy-longlegs Phalangium opilio, which bears a highly plesiomorphic arrangement of visual systems. We show that eyeless (ey) and twin of eyeless (toy) are expressed early in the developing head lobes of P. opilio, whereas sv is not. Gene silencing of ey, toy, and sv individually had no discernible effect on eye development. By contrast, double knockdown of ey and toy resulted in defects or loss in all three eye pairs of P. opilio. These data are consistent with a conserved role for Pax6 homologs in patterning both median and lateral visual systems across arthropods. Our results suggest that heterochronic shifts in expression, rather than changes in function, underlie the atypical dynamics of Pax genes in derived arachnid groups such as spiders.
Abstract The distribution of fitness effects (DFE) — describing how harmful, neutral, or beneficial new mutations are — is central to understanding how populations evolve. Although the DFE varies across genomes and species, it remains unclear which aspects of genomic organization drive this variation. Here, we inferred gene-level selective constraints across the genomes of Mus musculus castaneus , Drosophila melanogaster and Saccharomyces cerevisiae using a combination of population genetics and machine learning trained on diverse gene features. Many gene features were predictive of selective constraint, with conservation, gene structure, and expression being the most informative. These selective constraints delineated gene classes with distinct DFEs. Genes with higher connectivity and expression — features reflecting how many traits a gene influences — experienced stronger and less dispersed deleterious effects with increasing selective constraint. Between species, the rate of adaptation decreased with increasing organismal complexity, whereas across the genome it did not decrease monotonically with selective constraint, but tended to be higher at intermediate levels. While between-species comparisons of DFE parameters were less consistent with predictions of Fisher’s geometric model (FGM) based on organismal complexity, variation in DFE parameters across the genome aligned more closely with FGM when complexity was considered at the gene level. Our results suggest that gene-level complexity, captured by genomic feature proxies, provides a more informative definition of complexity for DFE variation than organism-level labels, and highlight the value of using gene features collectively to link genomic architecture, fitness landscapes, and patterns of molecular evolution.
Although strict maternal transmission of mitochondria is a general feature of animals for ensuring homogeneity in mitochondrial DNA (mtDNA) across generations, exceptions were reported in the recent past. For example, some extremely rare but spectacular cases of heteroplasmy and paternal transmission in humans have questioned the universal evolutionary principle. Hence, as an alternative, the Mega-NUMT concept was coined to explain this discovery and was thereafter partly proven to exist. This concept expands on the quite common transfer of mtDNA fragments to the nucleus (NUMTs) by considering the existence of multicopy mitochondrial nuclear insertions. Mega-NUMT reports are currently restricted to a few cases in animals, including humans. However, their detailed genomic organization, natural prevalence, and potential biological functions remain unclear. Here, we discovered that up to 60 full-sized mitochondrial genomes are integrated into the nuclear genome of the neotropical Drosophila paulistorum using long-read sequencing and in situ hybridization. The copies are organized in one cluster on chromosome 3, which we designated the "Dpau Mega-NUMT". Contrary to the rarity in humans, this Mega-NUMT is found at high prevalence (40%) in both laboratory lines and natural D. paulistorum populations of different semispecies. Additionally, the Mega-NUMT copies are phylogenetically separated from the current mitotypes of D. paulistorum. Together, these observations suggest long-term maintenance of the Mega-NUMT in nature. Hence, we propose that the Dpau Mega-NUMT may have been transferred to the nuclear genome before the D. paulistorum semispecies radiation and speculate based on these findings that it possibly is maintained at relatively high prevalence in nature by balancing selection or due to a yet undetermined function.
