EvoDevo

Hard cornification is the developmental process that produces the hardened epidermal structures that characterize tetrapod diversity, including scales, feathers, hair, and claws. This process relies on keratin proteins and additional reinforcing proteins that assemble, cross-link, and strengthen tissues to become mechanically resilient. Although the molecular composition of hard cornified tissues is known to vary among tetrapods, the program is least understood in amphibians. Within frogs, hard cornification occurs in the mouthparts of most tadpoles and in the nuptial pads, hindfoot claws, and foot tubercles of some adults. We characterized transcriptomic profiles of Bombina orientalis tadpoles at three developmental stages before, during, and after mouthpart cornification. We identified 25 keratin genes orthologous to amniote hard keratins in the B. bombina genome, 18 of which are upregulated in cornifying larval mouthparts. Furthermore, two of these hard keratin paralogs and an amphibian epidermal differentiation gene exhibit distinct spatial expression patterns within the jaw sheaths and keratodonts evidenced by in situ hybridization, suggesting they have discrete functional roles. The newly discovered diversity of the hard keratin repertoire in Bombina , and the concurrent expression of many of these paralogs in tadpole mouthparts, suggests that anuran hard cornification is much more complex than adult cornified traits thus far characterized in Xenopus frogs. Our results also challenge the notion that the diversification of keratins in Amphibia is primarily associated with the transition to more terrestrial habits, and may instead be shaped, in part, by the novel feeding apparatus of aquatic tadpoles.

Microbes have likely influenced animal development since its origin, yet how host and microbe partner across ontogeny remains poorly understood. Here we characterise a developmentally dynamic symbiosis in the marine sponge Amphimedon queenslandica by simultaneously profiling host and symbiont gene expression through embryonic, larval and metamorphic development. The transcriptional states of all three core, vertically inherited symbionts were strikingly stage-specific. Metabolic gene expression of the dominant symbiont, AqS1, peaked in brooded embryos, when host glycolytic and oxidative pentose phosphate pathway expression was lowest, and this peak was enriched for enzyme-encoding genes. In non-feeding larvae, AqS1 biosynthetic gene expression contracted across central carbon, vitamin and amino acid metabolism, while its import capacity increased. This contraction was selective. AqS1 genes for the final steps of arginine synthesis, which the host lacks, were minimally expressed in pre-competent larvae and upregulated at settlement together with host nitric oxide synthase, consistent with the known requirement for symbiont-derived arginine in nitric oxide-induced settlement. Histidine biosynthesis genes were instead maintained in the larva, coinciding with upregulation of host histidine-to-glutamate conversion. Host and symbiont nutrient transporters peaked in the same developmental stages more often than expected by chance. In the filter-feeding juvenile, host metabolism and efflux transport reached their maximum while the symbiont transcriptome changed little. These findings show that symbiont biosynthetic output tracks host developmental stage, and suggest that an inherited microbiome may contribute to successive stages of an animal life cycle through changes in its transcriptional activity rather than its composition.

The evolution of development is the primary mechanism by which morphological and functional diversity is generated. Comparing the developmental trajectories of single cells in entire embryos across species can reveal how gene expression is conserved and varies, as well as the molecular mechanisms underlying how cell- and tissue-specific changes in developmental regulation generate phenotypic differences. However, this perspective has not addressed the mechanisms of small-scale developmental microevolution, i.e, in closely-related species or even within a single species. The fish Astyanax mexicanus, which comes in two forms adapted to distinct environments - the eyed, river-dwelling morph and the eyeless, cave-adapted morph - is a remarkable model with which to investigate the evolution of gene regulation in a single species. Here, we generated and analyzed a single-nucleus RNA-seq atlas of the development of the two Astyanax morphs, from gastrula to swimming larva. Despite extensive changes in gene expression observed in the eight examined stages, for both ubiquitously-expressed and tissue-specific genes, and in both embryonic and extra-embryonic tissues, the temporal progression of cavefish and surface fish cells was similar in ordinal regression-based pseudotime models using 20-100 core genes. However, transcriptome-wide comparisons of gene expression waves revealed massive heterochronic shifts. In cavefish, some tissues had a majority of genes lagging behind in their transcriptional dynamics (e.g. the optic region), while in others, a majority of genes had accelerated dynamics (e.g. the mesoderm), or else, an equivalent number of genes were found in either category (e.g. the endoderm). Furthermore, cell fate acquisition and differentiation were variably affected in different cavefish lineages, with delays of ~6 hours in the retina, ~2 hours in the axial mesoderm and ~none in the extra-embryonic enveloping layer. Thus, cavefish embryos progress as a mosaic of different tissues, or modules, each following its own transcriptional tempo. Because gene expression differential regulation and time shifts were more prevalent in the early stages and gradually diminished throughout development, we propose that cavefish embryos initially undergo strong decanalisation before becoming re-canalized in line with the shared Astyanax developmental program.

Unlike most other heterotrophic protist lineages which synthesize glycogen, alveolates accumulate -glucan storage polysaccharides in the form of starch. Within Alveolata, ciliates do so by using a simple pathway analogous to that used for glycogen metabolism in eukaryotes. However, Myzozoa have evolved five additional enzymatic steps which are common to Dinoflagellata, Chromerids and Apicomplexa. Moreover, dinoflagellates have acquired seven additional enzymes that distinguish them from the Chromerids-Apicompexa lineages, while chromerids possess only a single enzyme not found in dinoflagellates. Such modifications are not found in other eukaryotic lineages save for those resulting directly from primary or secondary plastid endosymbiosis. The many differences affecting the starch pathway of Myzozoa can be explained by a single secondary or tertiary plastid endosymbiosis of a starch-containing alga followed by distinct compartmentalisation outcomes of starch metabolism in Dinoflagellata relative to chromerids and Apicomplexa.

Butterfly wing scales are intricately structured, cuticle-derived, extensions of single scale cells. Their highly ordered structures confer a range of functions, including producing structural color, which depends upon unknown molecular mechanisms controlling the secretion and ordering of an extracellular matrix (ECM). To find the genes required for patterning the ECM we compare scale nanostructure development between structurally colored blue scales and non-iridescent black scales on the forewings of the butterfly Heliconius sara using transmission electron microscopy, RNA-seq and proteomics. We first determined that structural differences between the two scale types are established from about half-way through pupal development and then investigated differences in relevant genes and proteins between scale types from this stage onwards. We show that knockouts of the genes coding for zona pellucida (ZP) domain proteins, miniature and trinity, and the cuticle protein gene, Cpr128 produce distinct effects on scale morphology and structure formation. In particular, some highly unusual scale morphologies are observed in miniature knockout individuals, suggesting a previously undescribed role of ZP domain proteins in structuring the ECM of scale cells and in mediating the mechanochemical interactions needed to produce the complex extracellular structures important for biological function in this system.

The origin of multicellular animals from unicellular ancestors remains a major evolutionary question. To address this transition, a reconstruction of the nature of the last unicellular ancestor of animals (LUAA) is key, and requires broad sampling of extant unicellular relatives, which together with animals form the clade Holozoa. Environmental surveys suggest that unicellular Holozoa diversity extends beyond the five groups with characterized members (Choanoflagellatea, Ichthyosporea, Filasterea, Corallochytrea, and Atreyea). However, the extent of the environmental diversity within and outside these groups remains unclear. To fill this gap, we here systematically re-evaluated Holozoa diversity using environmental 18S rDNA sequences compiled from updated datasets, including long-read metabarcoding surveys. We report nine uncharacterized lineages inside Holozoa (seven newly identified here), provisionally placed outside the described groups. We also uncovered a previously unreported diverse lineage deeper inside Opisthokonta, separate from Holozoa and Fungi. In addition, we identified 21 uncharacterized lineages within Choanoflagellatea and Ichthyosporea (eleven newly reported here). We used short-read metabarcoding datasets to characterize the environmental and geographical distributions of these lineages. Many are globally distributed and some can reach relative abundances comparable to those of the described groups, while others are restricted to specific environments. Our findings reveal substantial uncharacterized diversity missing from the cultured taxa used in comparative studies of animal origins. By placing this diversity in a phylogenetic and environmental context, our study will guide the targeted isolation efforts needed to broaden the taxon sampling available to reconstruct LUAA and unravel the unicellular-to-multicellular transition that gave rise to animals.

Hydractiniidae is a diverse family of colonial hydrozoans exhibiting substantial variation in colony organization, reproduction and life-cycle strategies, including species with and without a medusa stage. Several hydractiniids are also established model organisms, yet species boundaries and distributions remain incompletely resolved, limiting comparisons of reproductive and developmental traits across taxa. Here, we combine morphological and molecular data to reassess hydractiniid diversity, species identities, and life-cycle variation in material from the North Atlantic and Arctic Ocean. We integrate detailed observations of colonies and reproductive structures with DNA barcoding and mitochondrial genomes to evaluate species boundaries and provide molecular support for taxonomic identifications. Our results confirm 10 species in the study area, occurring from shallow waters to 2600 m depth and from coastal fjords to deep-sea hydrothermal vents. We further show that some historical species identifications and distribution records require reassessment. We establish a new genus for Arctic hydractiniid species (Bonneviactinia gen. nov.) and reassign two species to Schuchertinia. Mapping reproductive strategies onto the phylogeny reveals a broad pattern of increasing retention of medusa characters, from fixed sporosacs through reduced and fixed medusoids and non-liberated eumedusoids to fully developed, free-swimming feeding medusae, suggesting a progressive evolutionary trajectory in life-cycle complexity across Hydractiniidae. Furthermore, we clarify the identity of Podocoryna carnea and, through rearing experiments from polyp stage to medusae, characterize its morphological variation and developmental stages. By linking morphologically documented specimens and vouchers to genetic data, we establish reference resources that will facilitate future taxonomic, phylogenetic and biodiversity studies, including molecular surveys of marine communities. Together, these results demonstrate the value of integrative taxonomy for resolving hydractiniid diversity and provide a comparative foundation for investigating the evolution of reproductive strategies and life-cycle complexity in Medusozoa.

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

Ant larvae are blind and immobile, depending heavily on the adults for care in the form of feeding, grooming, and transport. However, they are not inactive colony members but play a central role in the organization of ant societies by modulating adult behavior and physiology. While adult ants have been studied extensively, we still know little about what sensory modalities larvae use to perceive their environment. Here, we provide a molecular and circuit-level characterization of the chemosensory system of the clonal raider ant larva. Combining scanning electron microscopy and confocal imaging, we show that dome sensilla in the maxillary and labial palps are innervated by odorant receptor (OR) neurons. A larger population of ionotropic receptor (IR) neurons innervate papilla sensilla across various regions of the larval head: the antennae, the labrum, the maxillary palps and galeae, as well as the labial palps and the ventral labium. This suggests that IRs, which can have various functions, from taste and olfaction to temperature and humidity sensing, constitute the predominant chemosensory modality in ant larvae. To investigate projections into the central brain, we imaged transgenic lines that fluorescently label OR- and IR-expressing neurons. We found that OR- and IR-expressing neurons in the labium and maxilla project along the labial and maxillary nerves through the subesophageal ganglion to the deutocerebrum, whereas IR-expressing neurons from the labrum project to the tritocerebrum and those from the antenna project to the posterior deutocerebrum. Together, our findings establish a structural and molecular framework to study larval chemosensation in ants.

Food availability in natural habitats is often unpredictable and drives the selection of adaptive strategies in organisms to survive food scarcity. However, the genetic and molecular mechanisms that enable animals to survive under such nutritional extremes are poorly understood. Naturally adapted organisms therefore provide powerful genetic systems for uncovering the mechanisms that maintain energy homeostasis under nutritional extremes. Here, we leveraged the cavefish Astyanax mexicanus to identify a naturally occurring Thr166Met missense mutation in the melanocortin 3 receptor (mc3r) that reprograms energy homeostasis to facilitate adaptation to nutrient deprivation. To determine its functional consequences, we introduced the mutation into zebrafish mc3r using CRISPR/Cas9 genome editing. We show that adult mc3r mutants exhibit increased body size, adiposity, and hyperglycemia relative to wild-type animals, recapitulating key metabolic features of Tinaja cavefish and identifying mc3r as the first causal gene behind cavefish hyperglycemia. Investigations for early developmental consequences of the mutation revealed accelerated yolk utilization in mutant zebrafish larvae and strikingly elevated metabolic rates relative to WT, thus, uncovering a previously unrecognized role for mc3r in regulating energy expenditure. Importantly, the phenotype was also observed in Tinaja cavefish revealing a novel cavefish phenotype and establishing causal role for mc3r in elevated metabolic rate phenotype of cavefish. Together, these findings reveal that the Thr166Met variant produces distinct physiological consequences across life stages, enhanced energy storage and hyperglycemia in adulthood despite elevated larval energy expenditure. We propose that increased larval metabolism may support the energetic demands of enhanced foraging, whereas increased adiposity and blood glucose later in life provide energy reserves during periods of nutrient scarcity. More broadly, our study demonstrates how a single naturally evolved coding variant can reshape energy homeostasis across development and contribute to adaptation to nutrient-poor environments.

The olfactory ventricle (OV), an anterior extension of the lateral ventricular system into the olfactory bulbs, is generally considered a transient developmental structure that closes during early postnatal life. However, this view largely derives from a limited number of laboratory models, and the evolutionary distribution and cellular organization of the adult OV remain poorly understood. Here, we combined a comparative survey of mammalian neuroanatomy with phylogenetic analyses and cellular characterization of the OV across species. We compiled anatomical data for 154 mammalian species spanning 26 orders and 80 families, revealing widespread persistence of a patent OV into adulthood. Phylogenetic analyses supported OV presence as the ancestral mammalian state and indicated that its absence is phylogenetically structured and consistent with repeated secondary losses. Chiroptera displayed multiple lineage-specific losses, whereas variation within Muridae extended to the intraspecific level, with contrasting OV persistence among Mus musculus and Rattus norvegicus populations. Comparative histological analyses in sheep, Peromyscus maniculatus, and CD1 mice revealed that, despite marked interspecific differences in OV morphology, the persistent ventricular region retains a conserved cellular organization characterized by progenitor- and glial-associated markers, proliferative cells, immature neuronal populations, and a differentiated apical/ciliary interface. Although these observations do not establish local neuronal production or demonstrate that the OV constitutes an autonomous neurogenic niche, their convergence across species indicates that the persistent adult OV is not simply an anatomically residual lumen but a specialized component of mammalian olfactory bulb organization. Together, our findings identify the OV as an ancestral and widely conserved feature of the mammalian brain and provide an evolutionary and cellular framework for investigating the developmental mechanisms controlling its persistence and its potential contribution to adult olfactory neurogenesis and plasticity.

Understanding how genomic architecture and demographic history interact to shape patterns of genomic divergence is central to explaining adaptive radiation and speciation. Here, we provide the first full sequence patterns of diversity and differentiation for Hydrophis sea snakes, using population sampling of three ecomorphologically distinct, coexisting species that diverged near-simultaneously within the last few million years (H. major, H. elegans and H. curtus). We found recent population expansions in all three species, with little evidence for pervasive interspecific gene flow. Genomic landscapes of differentiation were correlated across pairwise comparisons, with relative divergence associated with local reductions in nucleotide diversity but mostly decoupled from absolute divergence. Peaks of differentiation were concentrated in putative pericentromeric regions, consistent with linked selection in shared low-recombination regions. In contrast, a large region of low relative differentiation on chromosome 2 showed elevated diversity across all species and overlapped a prominent cluster of chemosensory vomeronasal type-2 receptor (V2R) genes. Overall, our results suggest that shared demographic history and linked selection shape a common genomic landscape in Hydrophis, while chemosensory receptor gene evolution may have contributed to ecological diversification during the land-to-sea transition of these unique marine reptiles.

Venom systems are key evolutionary innovations that have contributed to the ecological success of numerous animal lineages. In ants (Formicidae), venom exhibits an exceptional diversity in both chemical composition and delivery mechanisms. Here, we combine a synthesis of current knowledge on the evolution of ant venoms with new phylogenetic comparative analyses, integrating chemical, morphological, and phylogenetic data at the genus level. Comparative analyses across Formicidae support the hypothesis that peptide-rich venoms represent the ancestral condition in ants, as they are widely distributed across basal and derived lineages. Despite this ancestral state, we identify seven independent evolutionary transitions toward non-peptidic venom, occurring in major clades including Dolichoderinae, Formicinae, Stenammini, Solenopsidini, Crematogaster, Pheidole, and fungus-growing ants. Ancestral state reconstruction and tests of correlated evolution across 114 genera show that these seven shifts away from peptide-based venom were almost irreversible and tightly coupled with the loss of stinging capacity and a dietary shift away from predation. These transitions are consistently associated with profound morphological modifications of the venom apparatus, such as reduction or complete loss of the sting, or its transformation into non-injecting structures. Chemically, these systems are dominated by alkaloids, iridoids, formic acid, esters, and terpenoids. These repeated transitions reflect major ecological and evolutionary shifts. Lineages that rely less on individual predation and more on collective foraging or plant-based diets tend to exhibit reduced reliance on injectable peptide toxins. Instead, they favor metabolite-based venoms that can be co-opted from existing biochemical pathways and deployed in diverse ecological contexts. Conversely, peptide-rich venoms are retained in lineages subjected to strong selective pressures for rapid prey immobilization or defense against vertebrate predators. Overall, ants represent a unique model for understanding venom evolution, illustrating how shifts in chemistry, morphology, and ecological strategy can drive repeated and convergent innovations. Future integrative studies combining genomics, metabolomics, and functional assays will be essential to unravel the mechanisms underlying these transitions and their role in the extraordinary diversification of ants.

The production of calcified skeletons by stony corals is the cornerstone of reef ecosystems and the persistence of marine biodiversity. Corallimorpharians, sometimes described as naked corals, resemble stony corals in many respects but lack hardened skeletons. However, whether they are stony corals that secondarily lost their skeletons has been a subject of much debate. Here, we present a chromosome-level genome of the corallimorpharian Ricordea yuma and show that macrosynteny and microsynteny analyses provide strong support for corallimorpharians as the sister group to stony corals. This phylogenetic placement indicates that corallimorpharians were not ancestrally skeletonized and therefore occupy a critical outgroup position for reconstructing the origin of coral calcification. Guided by this phylogenetic context, we sequenced whole-polyp single-cell transcriptomes from R. yuma and the complex coral Galaxea fascicularis and found that the scleractinian skeleton evolved through the emergence of a derived calicoblast cell state in stony corals. A combination of comparative genomics and regeneration experiments in G. fascicularis show that the emergence of the calicoblast cell state was accompanied by the evolution of a biomineralization toolkit assembled from lineage-specific innovations and co-opted ancestral components. Although corallimorpharians retain some ancestral genes later incorporated into coral biomineralization, they lack a detectable calicoblast-like transcriptional state and many core calcification genes. Together, our findings indicate that coral calcification arose through coordinated changes in genome architecture, gene repertoire, and cell identity, highlighting this process as a major evolutionary transition driven by the integration of genomic and cellular innovations.

Energy and nutrient regulation in eukaryotes depends on AMP-activated protein kinase (AMPK) and target of rapamycin (TOR), yet their evolution is usually described by which components are present or absent. We surveyed 10 TOR- and AMPK-related targets across 210 eukaryotic species and paired that inventory with sequence- and structure-level measurements of the proteins that remain. Across 2100 species-by-target cells, 1255 were coded present, 683 absent and 162 not assessable, with repertoires differing among lineages. Phylogenetically corrected analyses recovered associations among a subset of component absences, especially within canonical complexes, although correlated non-detection in incomplete proteomes remains an alternative explanation. Sequence features varied substantially. In TOR, N-terminal HEAT-region divergence was associated with absence of several TOR-complex partners in discrete-state analyses. In AMPK, the activation-loop threonine was retained in every species with a readable site, while other features varied strongly: {beta} carbohydrate-binding-module conservation covaried with C-terminal length under the primary phylogenetic model, and AMPK{gamma} showed pronounced lineage-associated length variation. In the focal coccidian-group cohort, {gamma} sequences had a median length of 909 residues versus 396 in other sampled copies; the median was 924 among the nine sequences with a tree-based orthology determination, while two Eimeria sequences remain provisional candidates. Low primary nucleotide-contact-site identity was shared more broadly across sampled Apicomplexa. Together, these analyses reveal lineage-structured variation in TOR and AMPK at three levels: component repertoires, sequence features of retained proteins, and associations among components. Signaling architecture cannot be inferred from presence and absence alone.

Torpor is a physiological strategy used by which some species conserve energy by lowering metabolic rates and body temperatures. While mammalian hibernation is well-studied, the molecular mechanisms governing the rapid entry into avian torpor remain poorly understood. This study represents the first multi-tissue whole-transcriptomic analysis of torpor entry in an avian system. By combining real-time thermal imaging and respirometry with transcriptomic profiling across seven tissues, we identified the key molecular pathways orchestrating this transition in Anna's hummingbirds (Calypte anna). Our results reveal that avian torpor entry is characterised by a subtle transcriptomic change (<5% of the genome differentially expressed across tissues) compared to mammalian hibernation (10%-60%). Most of these genes are differentially expressed before torpor entry is complete, rather than tracking body temperature linearly. This indicates that active transcriptional reprogramming precedes and potentially drives the transition. Thus, hummingbird torpor entry is not a passive shutdown, but a highly regulated, tissue-specific process. Vital organs, such as the heart and lungs, remain transcriptionally stable to maintain essential functions, while the liver and gut exhibit extensive metabolic rewiring. We found evidence of global transcriptional and translational suppression, and cell-cycle arrest: the three major contributors to cellular energy budgets. We hypothesise that avian torpor entry is orchestrated through an arrest of apoptosis, a metabolic switch from carbohydrate to lipid utilisation, coupled with alterations in alternative splicing and circadian rhythm regulation. Finally, the counterintuitive upregulation of genes involved in mitochondrial metabolism suggests a preparation for rapid arousal despite the depressed metabolic state.

Aquaporin-10 (AQP10) is an aquaglyceroporin that transports small, uncharged molecules, such as water and glycerol. In some species, such as mice and cattle, the AQP10 gene (Aqp10) is pseudogenized. In rodents, pseudogenization of Aqp10 is limited to the Myomorpha suborder, while Aqp10 remains intact in other rodent lineages. The order Cetartiodactyla encompasses diverse species, including Suina, Tylopoda, Ruminantia, and Whippomorpha. The distribution of intact Aqp10 across the entire Cetartiodactyla and analyses estimating the timing of Aqp10 pseudogenization have not been sufficiently investigated. In this study, we identified species within Cetartiodactyla that retain intact Aqp10 and analyzed the detailed patterns of pseudogenization to elucidate the specifics of pseudogenization within this order. We collected Aqp10 loci by BLAST and synteny analyses of publicly available genome databases of 51 cetartiodactyl species from 17 families. Dot plot and alignment analyses revealed that seudogenization of Aqp10 occurred in 42 Cetartiodactyla species, including the Camelidae, warthog (Suidae), Antilocapridae, okapi (Giraffidae), Cervidae, Bovidae, Hippopotamidae, and Cetacea. In contrast, nine species retained an intact Aqp10: pig and babirusa (Suidae), peccary (Tayassuidae), mouse deeres (Tragulidae), giraffes (Giraffidae), and musk deers (Moschidae). The timing of pseudogenization was estimated by comparing patterns of exon deletions, frameshifts, and nonsense mutations in Aqp10 pseudogenes. RT-PCR analysis of cattle and sheep tissues revealed that these species express Aqp3 and Aqp7 in their intestines, but not Aqp10. Collectively, these findings indicate that Aqp10 pseudogenization occurred independently in multiple cetartiodactyl lineages and provide new insights into the evolutionary history of Aqp10 loss within this order.

Phenotypic extremes are scattered across the Tree of Life, suggesting that evolutionary constraints often limit their emergence. Yet striking departures from standard body plans, such as elongate snouts, have evolved convergently across deep time. We investigate extreme morphologies in two distantly related deep-sea fish clades: spikefishes (pufferfish relatives) and snipefishes (seahorse relatives). Using spikefishes as a study system, including all 24 living and fossil species, we integrated genome-wide phylogenomic data with a fossil and extant morphological character matrix, skull shape and dentition analyses from 64 CT scans, phenotypic and divergence matrices from >200 X-rays, functional manipulations of freshly collected and cleared-and-stained specimens, and formal convergence test across 300 percomorph species. We show that snout elongation evolved independently twice within spikefishes, reaching intermediate and extreme conditions along one predictable, ordered trajectory of additive skeletal rearrangements. Snout bones form a highly integrated, semi-autonomous module that channels evolutionary change along intrinsic lines of least resistance, coinciding with accelerated cranial evolution and expanded morphospaces in a clade with enhanced cranial mobility. Snipefishes converge on the same body plan through largely non-homologous elements and distinct linkage mechanics, except for near-identical vomer convergence. Extreme morphologies can emerge when phenotypic integration aligns with functional innovation, guiding evolution along repeatable trajectories.

Understanding the origins of anatomical innovations is central to elucidate the evolution of phenotypic diversity, as novel body plans often underpin the colonization of new ecological niches. The stomach, with its acid-secreting glands, evolved in the gnathostome ancestor and has been lost repeatedly. Claudin 18 (CLDN18) is a tight-junction protein that preserves gastric epithelial integrity against extreme H gradients and, in mammals, also acts in the lung through alternative first-exon isoforms. This dual role suggests that pleiotropy may shape the fate of CLDN18 after stomach loss. Here, we provide a detailed analysis of CLDN18 evolution across jawed vertebrates. CLDN18 originated in the gnathostome ancestor and is expressed in the stomach of elasmobranchs. We found no evidence of the gene in chimaeras or lungfishes, two additional agastric lineages, extending the loss of cldn18a previously described in agastric teleosts. Exon 1 architecture indicates a tandem duplication in the Euteleostomi ancestor, whereas stomach- and lung-associated isoforms are partitioned in most tetrapods examined. Monotremes, although agastric, retain only the lung-associated exon 1, consistent with lung function sustaining selection on the locus. Together, these findings reveal a complex evolutionary history involving gene loss, alternative splicing, tissue-specific regulation, and secondary exon loss, highlighting how pleiotropy and regulatory partitioning shaped the evolution of epithelia in jawed vertebrates.

A longstanding question in evolution is if similar morphologies shared by distantly related organisms facing similar environmental pressures belie similar functions. The carnivorous pitcher form has evolved in three distinct plant lineages and encompasses a suite of convergent morphological traits, providing a natural experiment to test hypothesized links between morphology and function. Multiple pitcher plant species possess fenestrations (i.e., small windows) that let light pass through the pitcher leaf. While research on one species, Nepenthes aristolochioides, demonstrates that fenestrations manipulate light to create false exits and increase prey capture, whether this convergent morphology serves this convergent function in other pitcher species remains unresolved. We explored the function of fenestrations in two additional species of pitcher plants, Nepenthes klossii and Cephalotus follicularis, where the morphology and function of fenestration were previously unexplored. We inspected micromorphology of pitcher structure, conducted an insect bioassay with live plants to test for links between morphology and function, and quantified traits associated with light manipulation from herbarium specimens. Both species exhibited fenestration and related pitcher morphology similar to N. aristolochioides. Yet our experiments did not reveal a significant effect of light passing through fenestrations on fly capture for these two species. Key differences between these species and the previously investigated species may be the presence of wax crystals and the absence of viscoelastic fluid. While future studies may uncover weak to moderate light manipulation function not detected in our study, our data do not support convergent function in these distantly related, morphologically similar species.