BMC Evolutionary Biology

Body size, through its links to various physiological traits, has often been hypothesized to influence evolutionary rates. Negative body size-rate correlations have been reported in the morphological or molecular evolution of several extant vertebrate groups, including mammals, birds, reptiles, and teleost fishes. In this study, we estimated body masses for 89 species of plesiosaurs, a clade of Mesozoic aquatic reptiles, and found that their body size evolution conforms to a three-regime Ornstein-Uhlenbeck process, indicative of constrained evolution. Rates of morphological evolution, inferred using the skyline fossilized birth-death process and the variable-rates model, show minimal support for a correlation with body size in this clade. Our results thus serve as a counterexample, suggesting that the negative body size-rate relationship is not a universal vertebrate pattern, but rather a trend restricted to certain lineages.

Arthropods exhibit an exceptional diversity of life histories, where developmental modes involve moulting stage progressions with changes ranging from the bare minimal to the dramatically transformative. While this variability drives many research questions aiming to understand evolutionary and developmental underpinnings of life history differences, it can complicate comparative analyses across taxa. However, this can be approached by applying a framework that defines metamorphosis as a post-embryonic stage progression characterised by substantial changes in morphology and adaptive landscape. Employing this framework with a phylogenomic dataset spanning 26 orders and encompassing four independently arising metamorphic lineages, we explore gene repertoire evolutionary dynamics potentially associated with metamorphosis in Pancrustacea. The approach contrasts gene family evolutionary dynamics inferred to have occurred in the last common ancestors of the metamorphic Insecta, Copepoda, Eucarida, and Thecostraca, with those of their sister lineages, as well as of descendent and ancestral nodes. The results reveal that the metamorphosis ancestors are characterised by an elevated number of gene family births and expansions. Expanded gene families share a set of commonly enriched biological processes across all metamorphosis ancestors, suggesting functional convergence by independent evolution of distinct gene families involved in embryonic and post-embryonic development and nervous system differentiation. Evolutionary modelling further highlights a subset of these families exhibiting signatures of adaptive, lineage-specific gene family size increases associated with metamorphic development. These families include genes implicated in neural and sensory development, segmentation, and moulting. These findings support a model of the evolution of pancrustacean metamorphosis where distinct gene families from a common functional toolkit expand and are co-opted into facilitating transitions to multi-phasic life cycles. This reframes the role of moulting in arthropod diversification to be recognised as an important reservoir of genetic change that can potentiate truly remarkable life history transitions.

Molecular phylogenetics based on primary sequence comparisons has been central to reconstructing protein evolution. However, structural evolution does not necessarily parallel sequence divergence, particularly in proteins combining ordered domains with intrinsically disordered regions (IDRs). Here, we introduce a quantitative secondary structure distance (S2D) metric that enables systematic comparison of protein secondary structure, including both ordered elements and IDRs. Using the MADS-box transcription factor family as a model, we show that structural divergence is domain-specific and only partially coupled to sequence-based phylogeny. Domain-resolved analyses reveal that the DNA-binding M domain remains structurally constrained, whereas the I and C domains exhibit extensive sequence divergence while retaining conserved intrinsic disorder. In contrast, the K domain contributes disproportionately to global structural variability. Integrating S2D with phylogenetic distance uncovers both convergent structural architectures among distantly related proteins and pronounced structural remodelling within closely related paralogs--patterns not evident from primary sequence comparisons alone. Residue-level analyses further demonstrate that the structural impact of mutation depends strongly on amino acid identity and does not scale directly with substitution frequency or conservation metrics. Together, these findings indicate that secondary structural evolution provides an additional dimension of protein diversification beyond sequence divergence. By integrating phylogenetic and structural distances, this framework offers a complementary approach to interpreting protein evolution, particularly in families containing mixtures of ordered domains and intrinsically disordered regions. Significance StatementEvolutionary relationships are typically inferred from primary sequence comparisons, yet structural evolution may follow different trajectories. By developing a quantitative measure of secondary structural divergence, we show that structural change within the MADS-box transcription factor family can both converge and diverge independently of sequence-based phylogeny. Intrinsically disordered regions exhibit extensive sequence divergence while retaining conserved disorder, whereas specific amino acid substitutions disproportionately reshape secondary structure. These findings demonstrate that evolutionary diversification operates through domain-specific structural modulation rather than uniform sequence divergence. Integrating structural and phylogenetic distances provides a complementary framework for interpreting protein evolution and reveals evolutionary patterns that remain hidden when relying on sequence comparisons alone.

SummaryEstimating deep-time diversification patterns and the establishment of extant biodiversity represent major challenges in macroevolution. Fossil record data provide essential information to address these topics, but their heterogeneous temporal and geographical distributions require using analytical approaches to process these data. Gardiner et al.1 (hereafter GEA) used a deep-learning model2 and a fossil-occurrences dataset3 to estimate neoselachian richness over the last 145 myr. Results and DiscussionGEA1 found that neoselachian diversity increased throughout the Cretaceous, was little impacted by the Cretaceous-Paleogene (K/Pg) mass extinction ([~]10% species loss), and peaked in the mid-Eocene but declined until the Present. While the Cretaceous increase in neoselachian richness is well known4, the other findings of GEA1 are at odds with current knowledge. With the exception of lamniform sharks, the perceived decrease in species richness in the recent past is most likely due to a drop in available fossil record data combined with difficulties in identifying extant species in the fossil record5. Similarly, all previous analyses of the impact of the K/Pg mass extinction on elasmobranch diversification have reported high extinction rates, a marked diversity drop, and delayed recovery6-7, despite heterogeneity across clades, ecology, and geographical distribution7. Taking the K/Pg as an example, we demonstrate that the discrepancies between GEA1s results and current consensus is most likely due to a combination of incomplete, unverified, and incorrect fossil-occurrence data with inappropriate methodology.

Uintatheres, mammals belonging to the extinct order Dinocerata, are among the most recognizable of all Paleogene ([~]66 - 23 Ma) organisms. Unmistakable for their bizarre skulls with multiple pairs of horns and saber-like upper canines, uintatheres have captivated paleontologists since the late nineteenth century. Since their initial discovery, uintatheres have been regarded as a classic example of dramatic sexual dimorphism in the fossil record, with males purported to be larger and possess more prominent horns and canines than females. However, the hypothesis that uintatheres were highly sexually dimorphic has never been formally tested. Here, I use traditional, linear morphometrics on a collection including most known skulls of Uintatherium anceps to quantify patterns of cranial variation within this taxon. Despite using a variety of traditional and novel statistical methods, I fail to detect any evidence of strong sexual dimorphism in Uintatherium. To verify my approach, I assembled a similarly sized dataset from Bison bison as an extant analog, and found strong, consistent evidence of sexual dimorphism. In light of these findings, as well as the current understanding of uintathere systematics and paleoecology, I argue that strong sexual dimorphism should not be treated as the null hypothesis for this clade.

The distribution of fitness effects (DFE) of mutations is a key determinant of both the efficacy of natural selection and the genetic load of populations. It also provides an indirect summary of the underlying fitness landscape, so the extent to which the DFE is conserved across species can offer insights into the invariance of these landscapes. Here, we infer the DFE of amino acid-changing mutations in 38 catarrhine primates using site frequency spectrum (SFS)-based methods. Our results are consistent with the nearly neutral theory, with interspecific differences primarily driven by variation in effective population size (Ne) and thus the efficacy of selection. Specifically, a one-order-of-magnitude increase in Ne is associated with an approximately 10% increase in the fraction of strongly deleterious mutations. Although DFE estimates exhibit clade-level clustering, this pattern largely reflects shared Ne rather than clade-specific effects, and in unscaled units, deleterious DFEs are broadly similar across species. These conclusions are robust to the choice of DFE parameterization, phylogenetic regression framework, and ancestral misidentification. Extending the analysis to non-additive dominance effects further shows that dominance is only weakly identifiable from the SFS and has minimal impact on comparative DFE inference. Finally, simulations demonstrate that demographic correction via nuisance parameters enables robust inference across a range of demographic scenarios.

Protein evolutionary rates vary widely across proteins and among sites within proteins, reflecting multiple molecular, cellular, and functional constraints. While protein-level properties, such as expression and essentiality, and site-level structural and functional constraints, are known to influence evolutionary rates, how these constraints combine across scales to determine site-specific evolutionary rates remains unclear. Moreover, because many protein features are strongly correlated, it is difficult to disentangle their individual contributions to evolutionary rate variance, and unified predictive models that integrate these properties are still lacking. Here, we use neural networks to predict protein evolutionary rates across multiple scales based on multiple molecular and cellular features. At the protein level, integrating molecular and cellular descriptors explains substantial variance in evolutionary rates across proteins in multiple eukaryotic species, including nearly 50% of the variance in humans and substantial fractions of the variance in other eukaryotic species. The model also allows us to identify proteins whose evolutionary rates deviate from expectations based on their molecular and cellular properties. At the site level, we found that structural and functional features explain a comparable fraction of the variance in relative evolutionary rates. By integrating protein-level and site-level predictors, the model explains up to 37% of the variance in site-specific evolutionary rates across proteins. Our analysis demonstrates that constraints at these two scales combine largely additively, with protein-level properties setting the overall evolutionary context and site-level properties shaping variation within proteins. Together, these results provide a quantitative framework for understanding protein evolution across biological scales.

Recent studies have shown that the Triassic stem-frog Triadobatrachus lacked the ability to jump off, but nonetheless had the forelimb strength to withstand the impact of landing from a jump. We propose a hypothesis to resolve this pseudoparadox: the strengthened forelimbs are former adaptations to forelimb-based digging that later made jumping possible by exaptation. Micro-CT data from a skeleton of Batropetes palatinus reveal thin cortical bone, confirming Batropetes as terrestrial. Combining adaptations to walking and digging, confirmed by statistical analyses, Batropetes is thought to have searched for food in leaf litter or topsoil. We interpret Batropetes as having used one forelimb at a time to shove leaf litter aside. Batropetes may thus represent an analog or possibly a homolog of the digging stage that preceded the origin of Salientia. We discuss the possibility of homology with the digging lifestyles of other "microsaurs" and other amphibians.

Synonymous mutations do not alter proteins but undergo natural selection in many species. For Drosophila melanogaster, reports on selection strength vary from undetectable to surprisingly strong. Here we apply a new method to estimate the population selection coefficient (2 Ns) for all 134 ordered pairs of synonymous codon changes. The method uses ratios of site frequency spectra (SFS) for codon changes to neutral changes, and does not depend on divergence data, or codon frequencies, and is relatively insensitive to demographic history. Results indicate that natural selection on synonymous codons is weak, with |2 Ns|<2.07 for all pairs of codons and |2 Ns|<1 for 64% of codon changes. Despite being derived solely from polymorphism data, codon fitness estimates are strongly correlated with observed codon frequencies. A selection-mutation-drift model based on our 2 Ns estimates accurately predicts codon usage, while a model based on mutation alone fails. Codon fitnesses correlate strongly with a measure of codon frequency covariation among genes, and codons with large frequency differences between high- and low-expression genes have high fitness values. Finally, we detect clear signs that selection favors codon changes that stabilize mRNA secondary structure. By avoiding divergence data, which have multiple sources of additional variance, and focusing solely on allele frequencies, a clear picture emerges of selection on codon usage. The convergence of multiple independent lines of evidence (codon frequencies, expression-dependent usage, covariation patterns, and mRNA structure) validates this polymorphism-based approach and provides a coherent framework for understanding selection on synonymous site evolution. Significance statementSynonymous mutations do not change proteins yet affect gene expression in multiple ways. Here we provide the first fitness estimates for each type of synonymous codon change. Using only allele frequencies--without divergence data, codon counts, or assumptions about preferred codons--we estimate the fitness of all 134 single-step synonymous codon changes in Drosophila melanogaster. Results reveal weak but non-negligible selection, with codon fitness values accurately predicting codon usage patterns, including expression-dependent biases, codon covariation across genes, and mRNA secondary structure effects. This work offers the first comprehensive fitness estimates for individual synonymous mutations, providing a direct population-level view of how subtle genetic changes shape molecular evolution and establishing a general strategy for quantifying weak selection using polymorphism data alone.

AbstractFossils and ancient crude pottery vessels function as physical "DNA containers," preserving oriDNA (original, in situ DNA) and accumulating eDNA (environmental DNA) over time. Ancient DNA (aDNA) serves as molecular fossils, chronicling evolutionaryhistory. Using "nano-affinitybead technology", we extracted and sequenced DNAfrom Lycoptera fossils in the Jehol region and a "round-bellied jar (Jar)" from the Erlitou period in Guangwu Town, yielding 236,545 primate sequences from the fossils and 86,908 from the pottery. We observed that the AFF value of DNA sequences negatively corre lates with species divergence time1, offering a quantitative measure of DNA preservation and host divergence. Some fragments distinct from modern genomic sequences, termed "intermediate DNAsequences" (IDS), have been identified. Many IDS exhibit an upper age limit, preserving characteristics of the last common ancestor (LCA) of the Hominidae and offering molecular insights into "Darwins puzzle". Among IDS, the SRRA subtype (Sequence Reversal and Rearrangement), identified in mRNA-coding exonic sequences, arises from the incorporation of a complementary antisense strand upstream of the sense strand, either adjacent to it or separated bya sequence interval. This introduces a novel post-transcriptional regulatory mechanism at the mRNA level, driven by SRRA, which presets hairpin structures and Indels in the UTR or CDS of exonic sequences, modulating gene variation. We propose: "SRRAs played a critical trial-and-error role in early Hominidae evolution, facilitating adaptive genomic changes, with some SRRA sequences later excised from exons", positioning SRRAas an evolutionary "genetic switch". Additionally, seven species -specific fragments (SSFs) of non-human primates (NHPs) linked to Asian Homo erectus were identified in the fossils, and pottery DNA reveale d sequences from tropical species (e.g., zebrass, oil palms), providing evidence of climate-driven local extinction and supporting paleo-ecological and paleo-environmental reconstruction. This method of analyzing aDNA from non-skeletal materials opens new avenues in paleontology, archaeology, and geology, guiding the tracing of ancient migration patterns and fossil searches. DNAfragments preserved within "DNA containers " exhibit an "old-few, new-many" turnover pattern, with many aDNA fragments displaying non-deamination. This evidence challenges prevailing perspectives, the authenticity criteria for aDNA, and the capabilities and scope of the traditional research method, necessitating a thorough reevaluation of the relevant knowledge framework. Furthermore, this study opens new opportunities for frontier research.

The fossil record is the most important tool in palaeosciences, so continually reviewing and attempting to reduce biases in its collection is necessary to curate the best possible record of past life on Earth. Biases in the fossil record are introduced through both biological processes and data collection. Here we have investigated the extent to which anthropocentric data collection has contributed to sampling bias in the assembly of the current fossil record. We have found that the current fossil record (represented in this study by the NOW database) is anthropocentrically biased, both temporally and spatially. Specifically, fossil locality density is higher in time periods when hominins are found, and in known hominin-bearing locations. This demonstrates the need to stop essentializing the narrative of human evolution in paleoscience to reduce bias in sampling of fossil localities.

The ratio of nonsynonymous to synonymous substitution rates ({omega}) constitutes a fundamental parameter for inferring adaptive protein evolution, predicated upon the assumption that synonymous substitutions are selectively inert. This premise, however, is increasingly untenable given evidence of selection acting on synonymous substitutions, driven by various biological processes such as translational efficiency and mRNA stability. In this study, we demonstrate that unmodelled synonymous selection introduces substantial bias into{omega} estimation, resulting in elevated false positive rates in tests for positive selection. To rectify this, we present BUSTED+S+MSS, a statistical framework incorporating Multiclass Synonymous Substitution (MSS) models into BUSTED, a method for detecting episodic selection. By partitioning synonymous codons into empirically derived rate classes, this approach accounts for global synonymous constraints. Application to five diverse clades--Drosophila, Caenorhabditis, Enterobacteria, Saccharomyces, and Primates--reveals that the inclusion of MSS components consistently improves model fit and reduces the proportion of genes inferred to be under positive selection. In Enterobacteria, genes retaining significance under the corrected model exhibit weaker constraint on synonymous substitutions (dSs), consistent with the hypothesis that unmodelled purifying selection drives spurious signals of adaptation. Furthermore, an information-theoretic analysis indicates that whilst site-specific variation (SRV) provides the primary correction, global synonymous rate variation (MSS) contributes a distinct second-order correction. In highly divergent alignments, these signals act in concert to improve model fit. The BUSTED+S+MSS framework, especially when coupled with an "error-sink" to absorb alignment artifacts, thus offers a computationally feasible means to disentangle adaptive nonsynonymous substitution from the confounding effects of synonymous constraint.

The rhesus macaque (Macaca mulatta) is one of the most widely used animal models in biomedical research, both as it resembles humans in key biological aspects and as it is characterized by a broad geographic range. Most of the individuals housed in U.S. research colonies have been sampled from either China or India, though notably the source population of these animals has significantly shifted over time. Given the substantial genetic and immunological differences between these populations, a deeper understanding of the underlying population structure is critically important for biomedical interpretation. Despite this, the demographic histories of these two populations remain poorly resolved. Here, we present an analysis of whole-genome, PacBio HiFi long-read sequencing data from ten unrelated individuals of each population, applying four related model- and non-model based demographic inference approaches, in order to reconstruct their ancestral history. We evaluated the fit of the subsequently estimated models against the empirical data, and incorporated underlying uncertainty in the mutation rates used for scaling. We inferred a well-fitting population history characterized by substantial structure between Chinese and Indian populations, with a split time [~]140,000 generations ago from an ancestral population of [~]65,000 individuals. We additionally inferred the subsequent history of size change within, and gene flow between, these populations, reaching the current estimated sizes of [~]220,000 individuals in the Chinese population and [~]14,000 individuals in the Indian population. The robust baseline demographic model established in this study will serve as a valuable resource for future research on this species, including for improved fine-scale recombination mapping, selection inference, and association studies.

BackgroundAlpha satellites, a superfamily of AT-rich tandem repeats, are the primary DNA component of centromeres in Platyrrhini and Catarrhini. Analyses of the human genome suggest that centromeres behave like biological ridges, with new alpha satellite families expanding at the centromere core, splitting and displacing older ones towards the pericentromeres. The Cercopithecini tribe, which displays an unusual chromosomal evolution involving multiple chromosomal fissions and centromere formations, represents a promising model to enhance our understanding of alpha satellite DNA evolutionary history. We previously applied targeted sequencing to centromere DNA from two distant species drawn from the Cercopithecini terrestrial and arboreal lineages, and characterized six alpha satellite families exhibiting varying mean sequence identities. MethodsCombining classical and molecular cytogenetics, we mapped the chromosomal distribution of these alpha satellite families across 13 Cercopithecini, one Papionini, and one Colobinae species. A nuclear marker-based phylogeny provided an evolutionary framework for interpretation. ResultsOur phylogeny identifies the terrestrial and arboreal lineages, and a newly designated swamp clade. We observed significant interspecies variations in alpha satellite patterns, including differences in presence/absence and distinct chromosomal distribution patterns (centromeric, pericentromeric, or subtelomeric). Families previously described as heterogeneous (83-87% mean sequence identity) exhibit a centromeric position in the swamp lineage, which is characterized by conserved karyotypes. In contrast, these families show a pericentromeric distribution in the terrestrial and arboreal lineages, replaced at the centromere core by more homogeneous families (95-98% mean sequence identity). In the arboreal clade, which is characterized by highly fissioned karyotypes, putative evolutionary new centromeres show a unique co-occurrence of highly homogeneous and heterogeneous families. Conclusion & ImplicationsWe propose a comprehensive evolutionary scenario for alpha satellite DNA in Cercopithecini, where younger families arise at the centromere core, shift toward the pericentromeres as they age, and eventually face extinction. Our study suggests that alpha satellite DNA and chromosomes evolve in an interdependent manner, with satellite diversification and displacement occurring in parallel with chromosome fissions and centromere repositioning. This comparative cytogenomic approach provides both support for the human-based evolutionary model for alpha satellite DNA and novel temporal insights into its diversification dynamics. Beyond evolutionary genomics, our findings highlight the potential of alpha satellite DNA to complement systematic studies in deciphering complex primate evolutionary histories.

Molecular evolution is often modeled as proceeding at consistent rates over time, with some deviations accommodated by relaxed molecular clock models. Here, we quantify the full extent of variability in branch-specific substitution rates across relatively well-calibrated eukaryotic phylogenies, confirming that punctuated change at the molecular level underlies evolution at the morphological level where punctuated dynamics are more commonly recognized. We also show how inferred substitution rates decrease systematically when measured across increasing time intervals. This scale-dependence persists across alternative clock models, calibration strategies, and prior assumptions, but disappears in simulated data evolved under a constant rate - suggesting that the phenomenon arises from time-varying substitution rates and reflects genuine properties of evolutionary histories rather than model artifacts. The observed pattern is analogous to the Sadler effect in sedimentary geology, where time-averaged rates decline with increasing measurement interval because sedimentation is episodic, with longer hiatuses occurring less frequently. The recognized scale-dependent bias in molecular evolution is not captured in current molecular clock models and significantly impacts inferences of evolutionary history, such as estimating the age of Metazoa and understanding the timing and nature of the Cambrian Explosion. Significance StatementRates of molecular evolution are central to reconstructing the history of life, yet they are often assumed to be approximately constant over sufficiently long timescales. By analyzing relatively well-dated evolutionary trees of eukaryotes, we show how inferred rates of genome change systematically decrease as the timescale of measurement increases. This pattern is analogous to a well-known phenomenon in sedimentary geology where apparent sedimentation rates decline over longer intervals due to episodic processes. Our results demonstrate how long-term variability in evolutionary rates similarly produces a significant scale-dependent bias which is overlooked in current evolutionary models. Recognizing and quantifying this effect is important for dating key evolutionary events, such as the origin of animals, and for understanding the cadence of evolutionary processes.

Antimicrobial peptides (AMPs) are key defence molecules of the innate immune system of plants and animals. Understanding the evolutionary origins of AMPs can help to explain how immune systems acquire novelty and vary in their defensive capabilities. However, AMPs evolve rapidly, and so the origins of similar AMPs across organisms is often unclear. Furthermore, false negatives due to low search sensitivity are common and can hinder confident annotations about true absences. Due to these difficulties, understanding whether similar AMP genes found in diverse organisms represent ancestral molecules or evolutionary novelties has been challenging. In this report, we present evidence of horizontal gene transfer (HGT) of the antifungal peptide gene Drosomycin across insects. We show that in Diptera, the presence of Drosomycin is restricted to the Melanogaster group and additionally the distant relative Drosophila busckii. We go on to recover Drosomycin genes in cockroaches (Blattodea), mantises (Mantodea), one katydid (Orthoptera), various beetles (Coleoptera), and a recently acquired pseudogenized Drosomycin locus in Liposcelis booklice (Psocodea), but no other insects. Explaining this diversity through shared ancestry requires at least 50 independent loss events, or just seven HGT events. Previous studies have suggested that similar AMPs found across divergent species reflect conservation from a common ancestor, or due to their small size, that they arose via convergent evolution resulting from pathogen-imposed selection. Our findings suggest horizontal gene transfer can be responsible for the presence of some AMP genes found scattered across the tree of life. By presenting a mechanism through which immune systems can acquire novelty, our study also suggests a possible explanation for certain lineage-specific competencies for defence against infectious disease. While loss of AMP genes is common in certain lineages, here we suggest gain of AMPs can occur just as suddenly.

Despite the coppery titi monkey (Plecturocebus cupreus) being a model system for the study of neurodevelopment and behavior, the evolutionary forces shaping observed levels and patterns of genetic variation in the species have remained poorly studied. In order to illuminate the pervasive eCects of purifying and background selection, we have fit a distribution of fitness eCects of newly arising exonic mutations, utilizing patterns of polymorphism and divergence based on a recently published high-quality genome assembly. To further characterize episodically acting selective processes, we additionally performed the first whole-genome scans for recent positive and balancing selection in this species, reducing false-positive rates by incorporating the demographic history of the population into an evolutionary null model. These scans identified a small number of biomedically-relevant genes with strong statistical support for having experienced recent selective sweeps or long-term balancing selection. In addition, we identified four genomic deletions bearing the signatures of balancing selection. Taken together, this study provides the first insights into patterns of persistent and episodic selective processes in this species.

Pleiotropic genes control multiple traits. This can result in evolutionary antagonism because adaptation that favors one trait can interfere with the function of another. While pleiotropic genes show statistical signatures of evolutionary constraint, many of them contain multiple domains that may evolve under different selective pressures. This could either strengthen or alleviate gene-level constraint. Here, we study pleiotropy within the immune system of six Drosophila species to disentangle gene and domain-level evolution. We hypothesized that the multifunctional nature of pleiotropic genes may promote within-gene variation in evolutionary rates of their domains compared to non-pleiotropic genes. Consistently, we found a greater within-gene variation in evolutionary rate among domains of pleiotropic genes than other gene classes, despite relatively low between-gene variation in evolutionary rates among pleiotropic genes. Non-pleiotropic genes, on the other hand, show a more heterogeneous selective pressure at the gene level. Regardless of pleiotropy status, domains within antiviral proteins show elevated evolutionary rates, while signaling protein domains show elevated ratios of radical to conservative amino acid substitutions, which likely have a significant effect on protein structure and function. Finally, an examination of plasticity in infection-induced gene expression responses across species revealed that non-pleiotropic genes with elevated evolutionary rates were also more likely to demonstrate variation in plasticity, but this relationship did not extend to pleiotropic genes. Overall, our results identify differences in evolutionary patterns across various biological levels (i.e., gene, domain, protein, and expression), showing that domain-specific evolution can potentially alleviate gene-level constraints.

The Down syndrome cell adhesion molecule (DSCAM) family, conserved across metazoans, plays key roles in neural development by mediating cell-cell recognition. Invertebrate Dscam evolved extensive molecular diversity through isoform diversification, whereas the vertebrate paralogs DSCAM and DSCAML1 followed a distinct evolutionary trajectory. However, how these vertebrate paralogs evolved after duplication, particularly with respect to functional divergence, remains poorly understood. Here, we investigated the evolutionary history and post-duplication divergence of these paralogs using phylogenetic, molecular evolutionary, sequence comparison, and transcriptomic analyses. Our phylogenetic analyses suggest an ancestral gene duplication predating the split between gnathostomes and cyclostomes. We found distinct patterns of selective constraint between the paralogs, particularly in the intracellular domain. In tetrapods, the intracellular domain of DSCAM showed strengthened purifying selection, whereas no comparable reinforcement was evident for DSCAML1, despite strong constraint in mammals. We also found distinct patterns of lineage- and site-specific positive selection between DSCAM and DSCAML1. Consistent with these evolutionary differences, comparative analysis of the intracellular domains revealed distinct repertoires of short linear motifs (SLiMs) predicted to mediate protein-protein interactions. Reanalysis of published transcriptomic data further suggested distinct downstream responses elicited by the intracellular domains of DSCAM and DSCAML1. Together, these findings suggest that post-duplication functional divergence of vertebrate DSCAM paralogs may have contributed to the evolution of molecular mechanisms underlying vertebrate neural development and circuit formation.

Evolutionary innovation is a key driver of the colonization of new environments and the adaptive radiations of major groups. Novel traits typically evolve through the modification of pre-existing characters but the genetic paths underlying their origin have been challenging to trace, and the general requirements for and relative order of different kinds of gene mutations have been difficult to assess. Here, we trace the genomic origins of four procoagulant venom toxins (factor X, factor V, group I phospholipase A2, and Kunitz-type toxins) that collectively underlie a novel, especially potent blood-clotting venom type in the recently evolved Australian brown snake and taipan clade. We discover evidence for a previously unknown fifth toxin, coagulation factor VII, and show that the toxins evolved through two distinct genetic paths. The factor X and factor V toxins evolved through the sequential de novo co-option of ancestral clotting factor proteins that entailed their heterotopic expression in the venom gland, the fixation of segmental duplications containing each locus, and subsequent gain-of-function mutations that rendered factor X and factor V constitutively active. In contrast, the phospholipase A2 and Kunitz-type toxins evolved by modifying the functions of neurotoxins that were part of the venom arsenal. Our findings support models in which innovative mutations in single-copy genes precede gene duplication in the evolution of novel proteins and offer a rare view into the genesis of a complex trait that has played a central role in a major adaptive radiation. Significance StatementThis study investigates how an entirely new blood-clotting venom type evolved during the recent radiation of Australias iconic venomous snakes. We traced the key genetic events that occurred on the evolutionary path to one of the worlds most potent venoms. We found that the novel venom activity evolved through the sequential co-option of multiple proteins of the snakes own blood-clotting system, followed by the modification of two venom neurotoxins into proteins with procoagulant activities. We suggest that these unique de novo gene co-options are seminal events that can unlock new ecological strategies, which in turn, may enable major adaptive radiations.