EvoDevo

The highly conserved body plan of nematodes makes members of this phylum excellent models to study cell type evolution. Early branching nematode lineages, mostly occupying aquatic habitats, usually contain caudal glands deployed for underwater attachment to a substrate, but have been thought to lack phasmid sensory organs, resulting in their historical classification as "Aphasmidia". With the transition to a terrestrial environment, nematodes lost caudal glands and gained phasmid sensory neurons. The supposed mutually exclusive existence of caudal glands and phasmids has led to the suggestion that phasmid neurons may have evolved from caudal glands. Here, we rule out this possibility through light and electron microscopical analysis of Mononchus aquaticus, a member of the early branching Dorylaimia lineage, showing that phasmid sensory neurons and caudal glands do coexist. This observation not only argues against a proposed cell type evolution scenario accompanying aquatic-to-terrestrial transitions but also indicates that the presence of phasmid sensory organs may have been an ancestral trait of the nematode phylum.

Amphibians develop both external and internal gills during ontogeny, offering an opportunity to investigate the developmental relationship between these positionally distinct respiratory organs. Although internal gills of vertebrates are widely accepted to arise from pharyngeal endoderm, external gills have long been regarded as purely ectodermal outgrowths, obscuring their relationship to other vertebrate gills. Here, we combine histological analysis with direct lineage tracing in the Mexican axolotl (Ambystoma mexicanum) and the African clawed frog (Xenopus laevis) to resolve the embryonic origin of amphibian gills. We show that the external gill develops as a continuous epithelial extension of the pharyngeal endoderm, which forms its basal epithelium and reaches the distal gill tip. In the frog, this extension remains continuous with the epithelium giving rise to the internal gills. Rather than representing separate epithelial structures, external and internal gills therefore arise from a shared epithelial domain of the pharyngeal endoderm. These findings resolve a longstanding question concerning the embryonic origin of amphibian gills and provide a developmental viewpoint for understanding how spatially diverse vertebrate gills can evolve through repeated modification of a conserved endodermal tissue.

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 origins and early diversification of intercellular signaling molecules in animals remain poorly understood because comparative data across basal metazoan lineages are limited. Cnidarians form the sister group to bilaterian animals, and characterizing their transmitter systems is critical to understanding how complex adaptations within integrative systems shape evolutionary trajectories. Although glutamate is a well-established transmitter in bilaterian animals, its role in cnidarians remains unclear, and information on its neuronal function and signaling is limited. For most studied cnidarians, glutamate has been suggested to be a non-neuronal signaling molecule. Here, using glutamate immunoreactivity (Glu IR) in eight hydrozoan species with distinct ecologies (Aequorea victoria, Eutonina indicans, Clytia gregaria, Bougainvillia principis, Euphysa flammea, Polyorchis penicillatus, Aglantha digitalis, Nanomia septata), we identified and visualized distinct populations of glutamate-immunoreactive (Glu-ir) cells, including nematocytes, neurons, and muscle cells. A broad diversity of Glu-ir nematocytes was found in all studied species. Glu-ir neural cells were found only in three species (Aequorea, Nanomia, and Aglantha); their morphology and localization were species-specific. In addition, some striated and smooth myoepithelial cells were found to be either Glu-ir or GABA-ir. We propose that both glutamatergic and GABAergic systems were independently recruited more than 3 times as neurotransmitters across cnidarians, and that these recruitments are fundamentally rooted in bioenergetic demands.

The nautilus (Nautilus sp.) is an early-branching cephalopods. It retains several conchiferan synapomorphies, including an external planispiral biomineralized shell. The shells complex structure allows it to withstand hydrostatic pressure, control buoyancy, and protect against external hazards. In this study, we comprehensively examined shell microstructures across different shell components and regions representing various ontogenetic stages in two adult museum shell specimens. We found that the nautilus shell is composed of five microstructural types (spherulitic, prismatic, nacreous, semi-prismatic, and irregularly oriented prismatic structures) organized into layered architectures within individual shell components and coordinated across the shell as an integrated system. Our observations highlight transitions between distinct microstructures within and across shell components and local variation within individual components such as the dorsal and ventral shell walls, suggesting that these patterns may contribute to shell strength and overall mechanical performance. Variation in caecum morphology suggests that this structure may be developmentally plastic and subject to relatively relaxed structural constraints. These findings show that the Nautilus shell is an integrated biomineral system in which diverse microstructures are organized across shell components to meet functional demands and provide the mechanical strength needed for survival.

The symbiosis with photosynthetic dinoflagellate algae enables corals to build and sustain reef ecosystems. Individual coral polyps hold algal symbionts in their epithelial endoderm cells and lose them under environmental stress, leading to coral bleaching. How the host integrates symbionts into its body plan is not well understood. Here, using a combination of high-resolution imaging, quantitative analysis, and environmental perturbations in the sea anemone Exaiptasia diaphana (Aiptasia) and reef-building coral Pocillopora damicornis, we uncover a spatial organisation of symbionts along the aboral-oral axis of cnidarian polyps that emerges under the long-range translocation of symbionts between host cells through a fluid-filled cavity. The symbiont distribution becomes specifically enriched in the tentacle bud endoderm during Aiptasia polyp morphogenesis. This pattern can form in darkness and with algae-sized inert spheres, suggesting an innate host-intrinsic mechanism. Symbiont-occupied host cells are mechanically constrained within the endoderm and thus unable to rearrange; instead, they go through cycles of symbiont expulsion and re-uptake via the host gastric cavity, with regionally biased rates of these behaviours providing a route to enrich symbionts in the tentacles. Symbiont organisation is remodelled under increased light in adult coral polyps, with a characteristic pattern of reduced tentacle enrichment, lateral clustering and retention in the body column emerging over a timescale of days. Together, our findings reveal that the spatial organisation of symbionts is dynamically regulated in cnidarian host tissues, a capacity that may shape both the establishment of symbiosis and its resilience under environmental change.

Webspinners (Insecta: Embioptera) are an unusual insect order that are known for their subsocial behavior and prolific silk-production. Due to their unique foreleg silk glands, and spider-like ability to produce silk throughout their entire life cycle, webspinners are hypothesized to have evolved silk independently from other arthropod lineages. To date, there are no reference-quality genomes available for the order, preventing the study of their silk gene origination and diversification. Here, we assembled PacBio HiFi reference genomes and characterized the silk genes present in two webspinner species, Aposthonia ceylonica and Oligotoma nigra. The genomes reveal multiple full-length copies of the primary Embioptera silk gene, e-fibroin, that have undergone both ancestral and recent gene duplications within the group. For both species, all e-fibroin paralogs show the presence of complex repeat units consisting of multiple exons and introns that are remarkably homogenized across each gene. We also used CT-scanning of the internal silk glands to provide details concerning the localization of silk production in foreleg tarsi, and interspecific morphology. Article summaryThis study introduces the first high-quality genomes for webspinners, enabling new research on silk for evolutionary biologists and materials scientists alike. The authors sequenced two embiopteran species, Aposthonia ceylonica and Oligotoma nigra, to compare silk genes and gland structure using micro-computed tomography, an imaging method that shows internal anatomy in detail. They found multiple copies of the primary silk gene in both species that likely arose from multiple duplication events at different evolutionary times. These silk genes exhibit unusual gene structure with hierarchically organized repeat units that are highly homogenized within a gene. The findings show that silk genes have a complex evolutionary history in webspinners and provide a foundation for studying silk diversity within the order, and in the broader context of insect silk.

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.

Despite glutamates widespread role as the dominant excitatory transmitter in vertebrate brains, the early evolution of glutamate and its recruitment into neural signaling remain largely unknown. The major limitation is the lack of information on its distribution in early-branching basal metazoans, such as ctenophores (comb jellies). Here, using glutamate immunoreactivity (IR) in two ctenophore species with distinct ecologies (Pleurobrachia bachei and Beroe abyssicola), we show that glutamate IR is present in subpopulations of neurons within the subepithelial neural network and in small groups of mesogleal neuron-like cells, and that it differentially labels some muscle fibers. Remarkably, we also observed an enriched glutamate-ir signal within the nuclei of subepithelial neurons in Beroe. However, glutamate expression levels are species-specific, suggesting a tight coupling of glutamate recruitment for neural communication with energetic demands.

Originating at the base of the bilaterian tree of life, the monoaminergic (MOA) system is a pivotal and evolutionarily conserved regulator of animal development and of responses to changing environmental conditions. Investigating the ontogeny of monoaminergic modulation in model systems such as marine bivalve molluscs is therefore particularly relevant, as their life cycle and developmental transitions are strongly influenced by environmental cues. Here, we characterized the spatio-temporal and tissue-specific expression of components of the MOA system during early larval development of the Mediterranean mussel Mytilus galloprovincialis using both time resolved transcriptomics and in situ Hybridization Chain Reaction (HCR). Our results identify serotonin and dopamine as the predominant and interconnected monoaminergic pathways deployed during early mussel development, with receptors, enzymes, and selective transporters broadly expressed across both neuronal and non-neuronal tissues. Notably, the expression of receptors preceding that of the corresponding biosynthetic enzymes indicates early, non-neuronal roles of monoaminergic signalling, supported by their localization in peripheral tissues such as ciliated epithelia. Altogether, These findings support the hypothesis that the MOA system acts as a pervasive and tightly regulated modulator of larval morphogenesis and could therefore play an evolutionary conserved role in mediating development and environmental plasticity in developing bilaterian organisms.

Regenerative capacity varies widely across flatworms (Platyhelminthes). Whereas catenulids, microstomids and planarians can regenerate a complete head de novo, other flatworms cannot. This striking diversity raises a longstanding evolutionary question: does whole-body regeneration represent an ancestral trait that was subsequently lost in multiple lineages, or did it evolve convergently? Addressing this question requires comparative analyses of the molecular mechanisms underlying regeneration across phylogenetically diverse flatworms. Here, we focus on Wnt signaling, a deeply conserved regulator of antero-posterior (A-P) patterning and a central determinant of head-versus-tail identity during planarian regeneration, to establish a mechanistic framework for such comparisons. Although Wnt signaling has been studied extensively in planarians and parasitic neodermatans, its evolution and deployment in other flatworm clades remain poorly characterized. To address this gap, we characterized the complement of Wnt signaling components in two early-diverging flatworm clades, Catenulida and Macrostomorpha, with particular emphasis on expression and function in the catenulid Stenostomum brevipharyngium. Phylogenetic analyses reveal the ancient loss of six Wnt families and one secreted Frizzled-related protein (sFRP) family in the last common ancestor of flatworms, followed by additional lineage-specific gene losses and expansions. Moreover, several Wnt pathway components display markedly divergent expression patterns between catenulids and other flatworms, while functional analyses indicate corresponding differences in their regenerative deployment. Together, our findings reveal a dynamic evolutionary history of the flatworm Wnt signaling toolkit and establish a comparative framework for testing whether the molecular circuitry underlying head regeneration is ancestrally conserved or has evolved independently in distinct flatworm lineages.

Colonial animals rely on agametic reproduction to generate repeated colony modules (zooids). Although zooid development has been extensively studied in fully colonial species, such as cnidarians, bryozoans, or tunicates, it provides limited insight into how and why animals transition from solitary asexual fission to colony formation. Here, we fill this gap by studying the dynamics of asexual development in the microscopic flatworm Stenostomum, which can alternate between asexual fission and colony-like linear chains, composed of several zooids connected tail-to-head. Combining ecological, developmental, physiological, and transcriptomic analyses, we demonstrate that in four species of Stenostomum, chain formation can be triggered by manipulating food availability. This ecological input changes the balance between somatic longitudinal growth and head morphogenesis rate, generating a transient modular organism without requiring a new developmental program. We found no evidence for division of labor among zooids or for enhanced predation avoidance of the worms in chains, indicating that chain formation might be a developmental byproduct without obvious adaptive value. Together, these findings suggest that a neutrally evolving developmental byproduct of food-modulated allometric growth may provide a mechanistic route from asexual reproduction to coloniality.

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.

Eph and ephrin genes encode receptor-ligand pairs that mediate contact-dependent cell signaling and are essential for nervous system development. However, less is known about the role of Ephrin signaling during adult tissue homeostasis and regeneration. Here, we investigated the role of Ephrin signaling in neural patterning in the planarian Schmidtea mediterranea. We discovered that RNAi against the Eph receptor EphR1 led to striking ectopic expression of the mechanosensory neuron markers pkd1L-2 and hmcn-1-L, without obvious disruption of the overall architecture of the central nervous system. To investigate the basis of this phenotype, we identified additional Eph receptor homologs and four putative ephrin ligands and assessed their function. An RNAi screen revealed that ephrin-1 phenocopies the defects of EphR1 RNAi. Temporal analyses of EphR1 and ephrin-1 inhibition revealed a progressive increase in pkd1L-2+ and hmcn-1-L+ cells, indicating an unappreciated role for Ephrin signaling in regulating neural patterning and cell number during adult tissue homeostasis. Together, these findings provide a framework for dissecting Ephrin-dependent mechanisms in adult tissue maintenance and regeneration.

Despite their currently scarce taxonomic diversity, rhinocerotoids were among the most evolutionarily successful clades of large herbivorous mammals throughout the Caenozoic. Within an array of morphological adaptations, the cranial horn represents the clades most remarkable and name-giving feature, imposing significant morpho-functional demands on the skull. In this paper, patterns and drivers of the rhinos cranial evolution were investigated for the first time by compiling a three-dimensional geometric morphometric dataset of living and extinct species. Multiple aspects of morphological evolution were explored using phylogenetic comparative methods, including craniofacial evolutionary allometry (CREA - tendency of larger species to have longer faces), phylogenetic constraints, and tempo of evolution. Cranial shape variation linked to horn presence and size evolved under a phylogenetically constrained framework, with a notable transition from hornless to horned species. Rhinos significantly deviated from CREA, likely due to diversity of cranial forms and proportions that evolved independently across the clade in response to varying horn morphologies and dietary habits. Hornless and horned rhinos exhibited similar rates of cranial evolution, and shape variation was obtained through multiple episodes of accelerated evolution. This highlights the role of morphological innovations and Caenozoic global cooling events in the emergence of phenotypic diversity.

Specialized suction discs are functional innovations that enable fishes to attach to diverse surfaces and resist hydrodynamic forces. The adhesive discs of clingfishes (Gobiesocidae) vary in size and shape, but it is unclear how ecological factors have influenced their morphological evolution. Here we analyzed the disc and skull shape of 74 clingfish species using micro-CT scanning and 3D geometric morphometrics to investigate the role of habitat and substrate use on patterns of diversification. We also present novel comparisons of adhesive performance for 10 clingfish species. Clingfish interface directly with their environment using their adhesive discs, but we found that the disc and skulls are evolutionary integrated and share similar responses to habitat. Transitions from coastal habitats to coral reefs promoted elevated rates of evolution and morphological disparity across the body, whereas transitions to freshwater did not. Concurrently, repeated shifts from living on hard substrates to softer substrates (i.e., macroalgae and seagrass) were associated with more constrained disc shapes, convergent morphologies, and differences in adhesive performance. We propose that habitat and substrate use make complementary contributions to skeletal diversification in clingfishes, but the adhesive system requires further investigation to disentangle the complex interactions between form and function.

Sea urchins are pivotal models in cellular and developmental biology, but their biphasic life cycle with an extended larval life limits the ability to study post-metamorphosis and adult characters. Here, we introduce a genomically-enabled model system, the tuxedo urchin Mespilia globulus, which has rapid access to late life stages in sea urchins. We describe how we cultured M. globulus in a landlocked marine facility, raised larvae under artificial conditions and closed their life cycle. We established the experimental tractability of M. globulus: we labelled transcripts by hybridization chain reaction (HCR), and knocked out pigmentation genes to produce albino larvae using CRISPR/Cas9. We generated chromosome-scale genome assemblies for two individuals representing both sexes and two color morphs (red and blue), and compared the organisation of the 21 chromosomes of M. globulus with that of other camarodont echinoid models. We determined that M. globulus showed a conservative gene repertoire lacking the gene family expansions seen in other camarodont sea urchins. We annotated the complement of genes associated with pigmentation, immune and nervous systems and profiled their expressions in tissues and organs. Finally, we surveyed sex-related regions in genomes using genome assemblies and resequencing data, finding no evidence of heteromorphic sex chromosomes in M. globulus. Our findings highlight the accessibility of this new sea urchin model for studying the metamorphosis and adult biology of sea urchins.

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.