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.

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 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.

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.

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.

Tissue regeneration requires careful allocation of metabolic resources, yet how organisms adjust this allocation in response to varying amounts of tissue loss remains poorly understood. Here, we show that the regenerative metabolic response is not fixed: the size of an injury regulates how glucose is allocated at both local and organism-wide levels. We first demonstrate that tail regeneration requires glucose metabolism in the axolotl (Ambystoma mexicanum), a salamander capable of regenerating centimetre-scale tissues. We then mapped glucose uptake in axolotls regenerating from small or large tail injuries using positron emission tomography/magnetic resonance imaging (PET/MRI) and the radiolabelled glucose analogue [18F]FDG. Glucose uptake was elevated in regenerating tails compared to uninjured tails. During early regeneration, larger injuries induced higher glucose uptake than smaller injuries, correlating with faster regenerative outgrowth. Larger injuries also increased glucose uptake in distant organs, indicating a systemic metabolic response. Together, our findings suggest that metabolic responses tuned to injury size underlie faithful tissue regeneration and establish PET/MRI as a powerful approach for studying whole-body metabolic dynamics in large regenerating vertebrates.

An animal's musculature must support its specific biomechanical needs, so muscle morphology and volume allocation may adapt when locomotor strategies diversify. We examined muscle size and morphology in two sister families of bombycoid moths, wild silkmoths (Saturniidae) and hawkmoths (Sphingidae), that have diverged in wingbeat frequency, wing morphology, and behavior. Although both families rely on the same muscles to power and steer flight, they may distribute muscle volume differently to prioritize distinct functions. We hypothesized that flight power muscle proportions are larger in hawkmoths and increase with wingbeat frequency, helping meet inertial power demands of high-frequency maneuverable flight. We also hypothesized that some individual muscles diverge in proportional volume and area to support distinct wing control strategies. To test our hypotheses, we took CT scans of twenty bombycoid species and quantified volumes and geometries of six flight muscle pairs. As expected, flight power muscle proportions positively correlate with wingbeat frequency and are generally greater in hawkmoths. Two of three steering muscles diverge substantially in relative volume and area between families. Most muscles exhibit greater length in silkmoths and greater cross-sectional area in hawkmoths. Finally, the dorsal oblique(DO) muscle diverges exceptionally in size and morphology, being highly developed in hawkmoths and smaller or absent in silkmoths. This unexpected difference supports the DO having an underappreciated role in flight control, possibly via shaping indirect strain propagation in the elastic thorax. We show that muscle volume distribution parallels bombycoids' divergent flight strategies, demonstrating how muscle allocation can adapt for specialized functional goals.

Resolving deep phylogenetic relationships requires integrating multiple lines of evidence, as distinct evolutionary forces shape signals from different genomic markers. Here, we investigate the systematics of the controversial APPD lineage (Anomiidae, Placunidae, Plicatulidae, and, by inference, Dimyidae) within Pectinida sensu lato using phylogenomic, comparative genomic, transcriptomic, proteomic, and morphological approaches. Our analyses consistently recover APPD as a monophyletic lineage sister to Limida and Pectinoidea, divergent at [~]428 Mya. With three novel high-quality genomes, extensive progressive chromosomal fusions demonstrate a reduction in chromosome number of the APPD lineage (6-13), compared with an ancestral 20 molluscan linkage groups (MLGs). Accompanied by extensive intrachromosomal gene-order scrambling, we identify one functional centromere in Placuna vitream flanked by two vestigial centromeric remnants on a single chromosome, providing a potential resource for investigating centromere inactivation and neocentromere formation. Mitochondrial genomes of APPD lineage exhibit unprecedented plasticity in translational decoding: Pododesmus employs the invertebrate mitochondrial code; Heteranomia employs +1 translational frameshifting to bypass in-frame TAG codons, whereas in Anomia, Enigmonia, Placuna, and Plicatulidae, TAA is reassigned to tyrosine and confirmed by proteomic evidence, which supports mitochondrial frameshifting in APPD lineage and defines a novel translation table for bivalves. Integrating phylogenetic distinctiveness, deep divergence, extreme karyotypic restructuring, unique mitochondrial features, and morphological diagnosability, we elevate the APPD lineage into Anomiida ord. nov. This revision resolves long-standing uncertainties for Pectinida sensu stricto and Limida, and establishes the APPD lineage as a valuable system for investigating chromosome fusion, centromere evolution, codon reassignment, and translational recoding. ClassificationBiological Sciences; Evolution SIGNIFICANCE STATEMENTWe have re-examined a controversial group of marine bivalves (Anomiidae, Placunidae, Plicatulidae, and Dimyidae). Our integrative approach shows that these animals split from scallops and their relatives more than 428 million years ago and have undergone drastic chromosomal fusions that reduced their chromosome number from 20 to as few as 6. Additionally, some species evolved unusual ways of reading their mitochondrial genetic code, either reassigning the stop codon to tyrosine or using +1 translational frameshifting to skip stop signals. The combination of deep evolutionary time and genomic divergence warrants recognizing them as a new order, Anomiida ord. nov. This work, as a case study, demonstrates how chromosome fusion and genetic code variation contribute to invertebrate diversity.

Most cockroaches and the termite Mastotermes darwiniensis are associated with Blattabacterium, an ancient obligate endosymbiont that participates in the nitrogen metabolism of its host. Blattabacterium has been vertically transmitted since it was acquired by the common ancestor of cockroaches and termites and was reportedly lost twice, once in the cockroach genus Nocticola and once in all termites except Mastotermes darwiniensis. Here, we acquired cockroach specimens spanning most of the cockroach phylogenetic tree to study Blattabacterium using shotgun sequencing. We found no traces of Blattabacterium in 64 specimens from ten independent lineages of cockroaches across three families: Blattellidae, Pseudophyllodromiidae, and Anaplectidae. The absence of Blattabacterium was confirmed with three PCR amplifications targeting the 16S and 23S ribosomal genes with primers specific to Blattabacterium. Notably, cockroaches lacking Blattabacterium were often infected by Rickettsia and Wolbachia, many of which were related to the mutualistic Wolbachia strain of Cimex lectularius, the common bed bug. These results indicate that cockroaches from Blattellidae, Pseudophyllodromiidae and Anaplectidae have lost their ancestral Blattabacterium endosymbiont at least ten times independently, with many of these losses possibly facilitated and compensated by new associations with mutualistic Wolbachia strains that may help provision the host with B vitamins.

The influences of the gut microbiome on animal physiology are well-documented, yet the developmental timing of microbial colonization and its long-term consequences remain poorly understood. In this study, we investigated how the timing of bacterial colonization during development affects transcriptional programming and phenotypic outcomes in adult Drosophila melanogaster reared on a common, rich diet. Using RNA-seq analysis on whole flies colonized either as newly hatched larvae or as newly eclosed adults, we observed minor but distinct transcriptional responses dependent on when flies were colonized. Both embryonic and adult colonization were associated with ~ 25 to ~ 200 differentially expressed genes compared to axenic controls, with the majority upregulated and enriched for immune-response genes, suggesting that colonization establishes a broader immune competence. However, only 10 genes showed persistent differential expression that was not normalized by introducing bacteria to adult axenic flies, including mitochondrial genes, the adipokinetic hormone (Adh), and a putative secreted neuropeptide. Overall, these findings suggest that Drosophila development on a rich diet is largely robust to the timing of bacterial colonization but that certain metabolic effects may occur.

Freshwater biodiversity is disproportionally high relative to the limited availability of freshwater habitats. This pattern is exemplified by freshwater fishes. Over 50% of freshwater fish species are concentrated in a single clade, Ostariophysi, including the 5000 species of minnows, carps, and loaches, the 4500 species of catfishes, and the over 2000 species of tetras, pirahnas, and characins. However, the relationships and ages of ostariophysans remain uncertain. We show that the initial diversification of ostariophysans involved only two freshwater invasions and was driven by the strikingly rapid origination of major crown clades, including Neotropical electric fishes, lutefishes, and multiple major living clades of catfishes, carps and minnows, and tetras and characins, within five million years of the Cretaceous-Paleogene mass extinction. This result is congruent with the record of well-preserved body fossils of ostariophysans, but contrasts with the controversial assignment of isolated teeth and bones from the Cretaceous to nested lineages of this set of freshwater fish radiations. Although we confirm that Alepocephaliformes, an obscure marine lineage, is the living sister to Ostariophysi, our results demonstrate that the former clade only recently invaded the deep ocean, a transition that involved the loss of structures essential for enhanced auditory capabilities in ostariophysans and the related herrings and anchovies. These results establish a surprisingly young age for the major lineages of living freshwater fishes.

Arcellidae is a family of testate amoebae within Arcellinida (Amoebozoa), comprising three recognized genera: Arcella, Galeripora, and Antarcella. Although species in the family have been studied for nearly two centuries, many historically described taxa and major morphological groups remain unsampled at the molecular level. Here, we provide a comprehensive review of Arcellidae and generate new cytochrome c oxidase subunit I (COI) sequences for arcellid species from Canadian peatlands, focusing on tall-shelled Arcella historically classified in section Altae sensu Deflandre. COI phylogenetic analyses recover a strongly supported monophyletic clade corresponding to North American representatives of Altae, providing the first molecular corroboration of this morphologically defined group. Within this clade, we redescribe Arcella leidyana based on modern material from Eeyou Istchee (Quebec). We further describe Galeripora purdoni sp. nov. from a calcareous fen in eastern Ontario, representing a novel terrestrial lineage within the genus, and redescribe Galeripora artocrea, which we transfer to Arcella based on congruent molecular and morphological evidence. Phylogenomic analyses of Arcellidae isolates from the Protist 10,000 Genomes Project reveal an additional deep lineage basal to Arcella and Galeripora. Together, these results highlight hidden diversity and demonstrate the importance of integrative approaches for resolving arcellid systematics and refining its classification.

The evolution of new traits can drive species diversification by facilitating the use of new resources, but environmental change may turn these same adaptations into liabilities.Trait loss is also often associated with the origin of new ecologies, but how losses modulate diversification remains unclear. The swim bladder allows ray-finned fishes to regulate their buoyancy and exploit ecosystems throughout the water column, yet this organ has been lost many times among species-rich lineages. Here, we show that timing and ecological context control the macroevolutionary effects of swim bladder loss. Many lineages of fishes lost the swim bladder over the last 66 million years as they specialized for benthic habitats where buoyancy regulation is unnecessary. Swim bladder loss enabled the descendants of these benthic fishes to diversify in the deep sea where extreme pressure makes its inflation untenable, and in the frigid, oxygen-saturated Southern Ocean, where loss of the oxygen delivery mechanisms required for swim bladder inflation carries little physiological cost. Yet, we detect a selective filter associated with swim bladder loss during extreme global warming 56 to 50 million years ago, when its absence limited the capacity of fishes to escape ecological disruptions on the ocean floor. These contrasting patterns explain how the loss of a complex trait promoted major ecological transitions without increasing overall diversification through deep time. As human activity drives rapid global warming, the evolutionary legacies of swim bladder loss may again shape the fate of marine fish diversity.

Placentation and lactation are hallmark mammalian reproductive traits and key processes facilitating the transfer of nutrients from the mother to the offspring. The basal mammalian lineage of egg-laying monotremes features a short gestation period supported by a simple placenta and an elongated lactation, similar to marsupials. Eutherian mammals by contrast evolved longer gestation facilitated by extensive placentation and shorter lactation. To understand the gene expression underpinning maternal-foetal nutrient transfer across mammals, we generated transcriptome datasets for the monotreme reproductive tract and mammary gland and compared this to expression in representative species from the other mammalian clades: marsupials (Tammar wallaby) and eutherians (mouse). This revealed extensive overlapping gene expression between the therian placenta and monotreme reproductive tract, including the first observation of retroviral gene expression in the echidna reproductive tract. Interestingly, we discovered that transport and development functions supporting foetal growth are expressed in echidna mammary gland and eutherian placenta, likely a signature of the shift from prolonged lactation to longer placentation. Together, this demonstrates that diverse modes of maternal-foetal nutrient transfer are underpinned by similar gene networks across mammals.

The end-Permian and end-Triassic mass extinctions, driven by massive catastrophic volcanism and prolonged hypoxia, fundamentally reshaped life on Earth. However, the genomic impacts of these two biodiversity crises on living organisms remain largely unknown. Here, we performed a genome-wide screening to identify accelerated evolved regions in the ancestral lineage of mammals that survived both extinction events. Nearly all (20/21) of these accelerated regions were located in protein-coding sequences, and 81% (17/21) were found to be associated with lung function. We further extended our analysis to three additional vertebrate lineages that experienced either one or both of the mass extinctions. Similar genomic signatures, involving accelerated evolution of lung-related genes, were also observed in these non-mammalian lineages. Collectively, these findings suggest that adaptation of lung-related genes to prolonged hypoxia may have occurred during the two mass extinction events, potentially driving a second evolutionary stage of the lung following the vertebrate transition to land.

Major transitions in evolution are associated with the loss of independent reproduction by formerly autonomous units. Termites provide a powerful system for studying this process because they exhibit diverse social systems in which worker developmental and reproductive potential declines with increasing colony-level organismality. However, the evolutionary sequence and developmental genetic basis of these transitions remain unresolved. Here, using comparative developmental transcriptomics across seven termite species that differ in workers' reproductive potential, we reconstructed the evolutionary history of termite social systems. We found that linear caste development, in which workers retain full reproductive potential, represents the ancestral state of termites. Bifurcated caste development, in which workers partially lose reproductive potential early in development, evolved independently multiple times, with two origins subsequently giving rise to superorganisms with unipotent, sterile workers. Ancestral gene regulatory network (GRN) reconstruction revealed that linear caste development evolved through retention of a juvenile-like worker state and co-option of a conserved developmental GRN characterizing hemimetabolous insect nymphal development, in which juvenile hormone, ecdysone and TGF-{beta} signaling pathways play central roles. The convergent evolution of bifurcated caste development repeatedly co-opted the GRN underlying linear caste development, heterochronically shifting its activity to earlier developmental stages. Finally, we found that the evolution of termite superorganisms involved somatization of the worker caste and co-option of a conserved endocrine GRN for terminal differentiation. Together, these findings uncovered repeated routes to reduced workers' reproductive potential through GRN co-option and highlight striking parallels between superorganism evolution in social insects and organismal evolution in metazoans.

Exaggerated sexually selected traits are known to be highly variable and their degree of expression is dependent on nutritional input. Yet the molecular mechanisms linking nutritional variation to phenotypic variation remain poorly understood. Here, we investigate how nutritional input shapes the development of male rear leg length, an exaggerated and highly variable trait in the water strider Microvelia longipes, using comparative transcriptomics and RNA interference gene knockdown experiments. We demonstrate that nutrition is the primary driver of gene expression variation, with male exaggerated rear legs exhibiting the highest number of nutrition-responsive genes. Moreover, the increase in morphological divergence between leg types or sex, which is systematically exacerbated by rich nutrition, is associated with increased number of leg-biased genes. These comparative analyses allowed us to identify BMP11 as specifically enriched in female and male rear legs. Knockdown of BMP11 abolishes nutritional plasticity in leg length only in males, positioning it as a key integrator of environmental, sex and developmental signals. Our findings reveal that transcriptional modulation provides a molecular interface between nutrition and trait exaggeration. This work advances our understanding of how environmental cues are translated into complex phenotypes and highlights the role of developmental plasticity as a substrate for evolutionary change.