Evolution & Development

The morphogenesis of the optic cup provides a robust system for studying how two apposed epithelial monolayers with distinct properties fold and stretch in a coordinated manner to form the primordial eye. While much research has been conducted on the temporal dynamics of retinal neuroepithelium invagination, the spatial organization and stretching of the retinal pigment epithelium has received less attention. The fish species Astyanax mexicanus offers a unique model to examine the mechanisms of optic tissue morphogenesis through a comparative lens, as it exhibits natural variation in eye development between its river-dwelling and cave-adapted morphs. Using quantitative 3D imaging of optic cups from both morphs, we found that RPE morphogenesis involves transient, graded, and anisotropic cell stretching that patterns the epithelium during optic cup shaping. Analyses of RPE nuclear spacing and cell morphology showed that tissue stretching gradually increases along the proximo-distal axis, suggesting maximal tension in the elongated distal RPE cells aligned along the optic cup meridians. Furthermore, nuclear volumes and apical surface areas of RPE cells scaled spatially along the same axis, independently of endoreplication. In the cavefish natural mutant, RPE expansion was delayed by over six hours and proximal stretching exhibited altered isotropy, indicative of disrupted temporal coordination and suggesting modified mechanical constraints. These results demonstrate that RPE morphogenesis is a highly heterogeneous process from a spatiotemporal perspective, offering new insights into the study of the biomechanical principles of eye development in vertebrates. Summary statementThis study reveals the emergence of cell morphology gradients within the retinal pigment epithelium during morphogenesis of the eye in two distinct populations of the same species of fish.

Conserved dorsoventral patterning systems have been proposed as evidence for a common evolutionary origin of centralized nervous systems in Bilateria, yet functional evidence outside vertebrates and arthropods remains limited. Here, we investigated the role of pax6 in the annelid Platynereis dumerilii using a mutant carrying a 61 bp deletion in the paired-domain coding region. Loss of pax6 disrupted ventral neuroectodermal patterning at 34 hpf, causing a shift in nk2.2 expression, narrowing of the nk6 domain, and downregulation of pax3/7, while msx expression remained largely unaffected. These early patterning defects were followed by selective neuronal abnormalities at 48 hpf, including displacement of TrpH-positive serotonergic cells and loss of posterior hb9-positive motoneuron domains. By 6 dpf, additional defects were observed in TrpH, ChAT, VAChT, and nk2.2 expression, accompanied by severe disruption of ventral nerve cord morphology and loss of the characteristic rope-ladder architecture. Together, these findings identify pax6 as a key regulator linking dorsoventral progenitor patterning, neuronal subtype specification, and nervous system morphogenesis in Platynereis. Our results provide functional evidence that the conserved dorsoventral patterning network plays an essential role in annelid ventral nerve cord development and support the view that important components of bilaterian nervous system patterning predate the divergence of major animal lineages.

The zebra mussel (Dreissena polymorpha) is an invasive species that causes extensive economic and ecological damage. Here, we identify and characterize the key components of the small RNA (sRNA) and RNA interference (RNAi) pathways in zebra mussels. Like other mollusks, zebra mussels have extensive microRNA (miRNA) and Piwi-interacting RNA (piRNA) machinery but lack or have modified canonical factors needed to produce small interfering RNA (siRNA). Specifically, the zebra mussel Dicer sequence displays substitutions in the conserved DEAD box motif that is required for substrate processivity, and this organism also lacks some attendant accessory factors such as R2D2. We sequenced the small RNA found in both isolated somatic tissue (adductor muscle) and whole animals (including germline), and identified both conserved and novel miRNA and diverse piRNA sequences, but few endogenous siRNAs. To determine whether their remaining sRNA machinery could still be co-opted to initiate gene silencing, we injected dsRNA targeting several genes into zebra mussel adductor muscle. The injected rpn8-targeting dsRNA reduced rpn8 mRNA levels and was processed into sRNA that resemble endogenous miRNAs and piRNAs. The levels of both sRNA types correlated with mRNA knockdown, suggesting that they may act together to initiate RNAi as seen elsewhere. dsRNA targeting other genes produced variable results suggesting that particular criteria may be needed to trigger an RNAi response in this assay. Our results characterize endogenous sRNA pathways in zebra mussels, establish that dsRNA can induce RNAi, and lay the groundwork for further optimizations to establish RNAi-based genetic manipulation tools for this damaging invasive species.

In many insect species, mating is coordinated through multimodal signaling, yet less obvious channels are often overlooked. In the burying beetle Nicrophorus vespilloides, chemical communication is well-documented, but the role of substrate-borne vibrational signals (stridulations) during courtship remains unknown. We investigated whether stridulation is essential for mating success through two sets of experiments. First, we found a positive correlation between the frequency of stridulations and both the number and duration of copulation events. Second, we employed a silencing experiment to test the necessity of these signals by silencing males, females, or both partners. We found no significant differences between silenced and control groups regarding the frequency or duration of physical contact and mounting events, suggesting that stridulation is not required for mate recognition or the initiation of courtship. However, the proportion of successful copulations relative to mounting events was significantly lower when females were silenced. These results suggest that while N. vespilloides relies on a redundant multimodal system that likely utilizes chemical cues to initiate mating, vibrational signals, particularly from the female, may play a critical role in facilitating successful copulation. This study provides the first evidence for the role of stridulation in the mating behavior of N. vespilloides and highlights the potential for female-mediated vibrational signaling in burying beetle courtship.

Gene regulatory divergence has emerged as a key feature in speciation, influencing gene expression differences that accumulate between diverging populations. Transcriptional regulation, mediated by cis- and trans-acting factors, modulates diverse developmental processes and is responsible for distinct species-specific gene expression profiles. Within interspecies hybrid individuals, negative interactions between divergent cis- and trans-acting factors can lead to gene misregulation and hybrid dysfunction at the organismal level. Such gene regulatory mismatch might disproportionately impact sex-biased and tissue-biased gene regulatory networks due to their unique selective pressures. To address these issues, we investigated the role of regulatory divergence in asymmetric hybrid incompatibility between sister species of Caenorhabditis nematodes (C. remanei, C. latens) by analyzing gene expression of reciprocal hybrids for each sex and key tissue types. Despite severe hybrid male sterility, hybrid males showed less misexpression of sex-biased genes than hybrid females, suggesting that the organismal phenotypic outputs of male-biased gene regulatory networks are more vulnerable to disruption than female-biased genetic networks. Additionally, we found more genes associated with cis- than trans-regulatory divergence, supporting the notion of a disproportionate role for cis-regulatory divergence between species. Moreover, we document extensive cis-trans compensatory X-linked regulatory divergence specifically from male transcriptomes, indicating distinct molecular evolutionary outcomes of stabilizing selection on regulatory controls in males and females. These insights derived from asymmetric hybrid misexpression expand our understanding of the evolution of sex-biased gene regulation in the face of stabilizing selection and identify candidate genes contributing to Caenorhabditis post-zygotic reproductive isolation.

The evolution of flippers for wing-powered diving in penguins is a striking example of tetrapod limb specialization. The modern penguin flipper is structurally reinforced by a characteristic dorsoventral flattening of the long bones accompanied by a reduction in distal forelimb musculature, features which emerged convergently in flightless diving birds and aquatic mammals. While an extensive fossil record informs the morphological sequence through which these changes occurred, the evolutionary pressures and developmental mechanisms underlying these modifications are unknown. We find that in avian and mammalian forelimbs, a flattened bone morphology only emerged in aquatic lineages that lost ancestral modes of locomotion, including in flightless diving birds, pinnipeds, and cetaceans. Using penguin embryos as an accessible model for investigating flipper development, we demonstrate that early patterning of forelimb musculoskeletal morphology is similar to that seen in forelimbs of non-aquatic birds. Instead, later modifications of gene expression and cell and tissue behaviors underlie flipper phenotypes. Thus, we find that in the early penguin forelimb, the initial cues that pattern the muscle do not differ from other avian species, however late embryonic changes in proliferation result in dramatic reduction of muscle. Likewise, forelimb bones in penguins initially have similar cross-sectional proportions to those in flighted birds. The shape of these bones is, however, remodeled late in embryonic development through a process that shares molecular hallmarks with bone ridge formation at tendon attachment sites. In these bones, ridge-forming tissue initiates at the ends of the bones (the epiphyses) and extends into tendon-like connective tissue along the lateral edges of the bone, widening the long bones along the anterior-posterior axis and producing a flattened bone. Using spatial transcriptomics and comparative genomic tools we determine that differentially expressed genes between the ridge-forming tissues and long bone cartilage are significantly enriched for signals of selection in the penguin lineage and that these genes may also be convergently evolving in marine mammals. Together, these data show that the evolution of musculoskeletal morphology in the penguin flipper occurred through expansion or novel deployment of molecular programs typically associated with tendon-attachment sites during late embryonic development.

Extremely elongate necks have convergently evolved in several amniote lineages, including both aquatic and terrestrial forms (Fig. 1). The development of such a feature brings with it advantages in obtaining food items, but also biomechanical challenges, such as flexibility, stability, lift, and inertia. In Tanystropheus, a particularly long-necked Triassic archosauromorph, the neck is composed of only 13, mostly extraordinarily elongated and slender cervical vertebrae and accompanying rod-like, overlapping ribs, making it arguably the most extreme example of neck elongation in tetrapod evolution (Fig. 1;1-6). Understanding the function of this remarkable neck provides insights into the limits of neck elongation in amniotes and the evolution of morphological novelties in Triassic reptiles. Here we present the first quantitative biomechanical analysis of the Tanystropheus neck using a digital model based on three-dimensionally preserved bones. We assessed its range of motion (ROM) and performed finite element analysis (FEA) on the individual cervical ribs and the neck model in different configurations. Our results indicate that the neck of Tanystropheus was not extremely stiff, as previously postulated, and the ribs likely did not impair its movements. They transferred tensile forces towards the base of the neck, similar to what hypothesized for sauropods7. This study elucidates the bauplan of an extremely specialized animal and brings us closer to understanding the patterns of achieving neck elongation in vertebrates.

In contrast to many other developing systems, in which axon pathfinding and synaptogenesis are separated in time, the pioneering axon of the individually identifiable caudal primary motor neuron in embryonic zebrafish forms en passant synapses during its stereotypical ventral growth. How simultaneous synaptic differentiation and axon pathfinding are coordinated is not fully understood. Here we ask what the role of the tac1 gene, coding for the synaptic tachykinin neuropeptides, is in this unique axon differentiation process. The gene is expressed during axon outgrowth and its disruption results in increased branch length of CaP axons and subtle morphological defects of the pre-synapse. These abnormalities are accompanied by a robust [~]1.5-fold increase in motor neuron activity and in spontaneous early contractions in tac1-deficient embryos. Furthermore, pharmacological inhibition of the tachykinin receptor (Tacr1) leads to altered CaP axonal morphology, mimicking the axonal phenotype observed in tac1-deficient zebrafish. These findings suggest that tachykinin neuropeptides modulate formation and activity of en passant synapses and prevent aberrant axon branching during growth of zebrafish motor axons. HIGHLIGHTS- tac1 refines CaP primary motor axon development in zebrafish - Loss of tac1 disrupts presynaptic maturation at the horizontal myoseptum - tac1 mutants show elevated motor neuron activity and spontaneous contractions

The origin of novel phenotypes can influence access to new ecological resources, which may have positive, neutral, or negative effects on subsequent phenotypic diversification. In this study, we tested the macroevolutionary consequences of a pair of putative functional innovations occurring in deep-sea fishes of the order Stomiiformes. Integrating phylogenetic comparative methods, micro-CT scans, and external body measurements, we recover a mosaic of diversification trends associated with these innovations. We found some evidence for elevated evolutionary rates in tooth morphology associated with the predatory dragonfishes, which possess a gap between their vertebral column and skull that exposes the notochord and enables neck-like flexibility. However, a second novelty building upon the first, a functional neck joint enabling extreme cranial kinesis, was linked to faster rates of skull evolution. Our results suggest that innovations that help shift ecological roles and overcome functional constraints related to those roles, like gape-limitation in prey depauperate habitats, may play an important role in promoting phenotypic diversification. This work builds on a growing body of evidence highlighting how the deep sea promotes phenotypic diversity, generating the extreme forms that are celebrated by scientists and the public alike.

Photoreceptor outer segments are sensory cilia whose maintenance depends on a balance between basal disc renewal and tip shedding, controlled by intraflagellar transport and axonemal microtubule organization. Microtubule plus-end proteins regulate microtubule dynamics and are strong candidates for roles in this process. In this study, mCherry-tagged EB1, EB3, and DCX were overexpressed in zebrafish (Danio rerio) cone photoreceptors under a cone-specific promoter. Eyes were examined at 5 and 10 dpf, and eyecup depth, diameter, and cone photoreceptor area were quantified relative to uninjected controls. At 5 dpf, all three constructs produced eyes indistinguishable from those of controls. By 10 dpf, all three constructs significantly increased eye cup depth and cone photoreceptor area. EB1 and DCX also significantly increased eye cup diameter. EB1 and, more severely, EB3 also caused retinal holes, mainly in the retinal pigment epithelium and at the outer nuclear/outer plexiform layer, along with misshapen cells near the inner plexiform layer. DXC did not cause retinal holes, but, like EB1 and EB3, produced enlarged, bulbous cone outer segments. The results show that overexpression of any of the three +TIPs results in a similar eye and photoreceptor overgrowth phenotype, while also producing construct-specific defects: EB1 and EB3 disrupt the broader retinal architecture, whereas DCX produces enlarged eyes. The shared outer segment hypertrophy suggests an imbalance between cargo delivery at the basal end and shedding of the distal tips. The organomegaly may reflect altered progenitor signaling in the ciliary marginal zone.

The hallmark of sessile adult ascidians is a vase-like shape with a single oral and atrial siphon. Ciona, however, develops two atrial siphons after metamorphosis, which subsequently fuse into one. The mechanisms underlying this fusion are unknown. This study suggests that Hox2 controls this process. Hox2-knockout animals using Transcription-Activator-Like Effector Nuclease (TALEN) retain two atrial siphons throughout their lives. During normal fusion, epidermal cells between the siphons flatten along the anterior-posterior axis. This cellular flattening does not occur in Hox2-knockout animals, suggesting that the shape change in the epidermal cells produces tension, allowing the atrial siphon openings to converge at the midline for fusion. Hox2-knockout animals lack cupular organs, which are suspected hydrodynamic sensors in the internal epithelium of the fused atrial siphon and on the sperm duct. Among several knockout attempts, atrial siphon fusion was reproduced by only one TALEN pair, suggesting that this phenotype is driven by a mutation having a broader effect than those abolishing protein function. Many ascidians, unlike Ciona, develop a single atrial siphon shortly after metamorphosis. Our findings suggest that a phylogenetically conserved gene, Hox2, establishes this group-specific atrial siphon formation mechanism in Ciona.

Brain size and structure are hypothesized to be adaptively designed to satisfy the behavioral requirements of securing food and living socially. The importance of these socioecological and sociobiological selective forces in brain evolution is constantly debated. Socioecological divergence is striking in the Neotropical ant genus Neoponera: N. apicalis is a generalist solitary predator forming small colonies of ~100 whereas N. commutata colonies are approximately 10 times as large and workers pheromonally organize cooperatively raids only on Syntermes termite colonies. We interspecifically compared the size and structure of the compound eyes, size and number of antennal glomeruli, mosaic brain scaling and synaptic processing (microglomeruli-MG). Our results indicate that N. apicalis workers have a larger number of ommatidia, antennal lobe glomeruli, and allometrically larger antennal and optic lobes than N. commutata. These sensory traits were associated with differences in higher-order processing architectures in the mushroom body (MB) microglomeruli (MG). N. commutata workers had an allometrically larger MB, perhaps due to their socially complex chemical foraging communication, although MG density in N. apicalis was higher in both the MB lip and collar, regions associated with processing olfactory and visual information, respectively. The increase in MG density in N. apicalis may be associated with higher demands for navigation, learning, and memory, as well as a higher density of antennal lobe glomeruli to support prey odor discrimination. In contrast, N. commutata workers had larger ommatidia and antennal lobe glomeruli. Larger ommatidia correlate with their diurnal/nocturnal habits and a larger MB Our findings indicate that differences in behavioral performance demands associated with socioecological differentiation are reflected in variation in visual and olfactory system structure, brain size, mosaicism, and synaptic organization. Our results support both social and ecological brain hypothesis as drivers of mosaic brain evolution.

The number of chromosome sets per nucleus is a fundamental trait, but why this number is nearly always two for multicellular eukaryotes remains unclear. Chromosomes are made of nucleic acids, which possess abundant phosphorus (P). Therefore, producing new chromosomes, as well as generating new cells and organismal growth, demands substantial phosphorus. Yet, because P is often limiting in nature, P availability could influence the prevalence of diploidy versus polyploidy. Here, we compare growth rates of diploid and triploid Potamopyrgus antipodarum, a freshwater snail, relative to P availability. Because diploid P. antipodarum are obligately sexual while obligately asexual individuals are polyploid, costs associated with sensitivity to P limitation in polyploids could also help explain the maintenance of sexual P. antipodarum. We raised juvenile diploid and triploid snails on either P-adequate or P-deficient diets and found that independent of P availability, juvenile triploid asexual snails grew faster and harbored higher P content as adults than sexual diploid conspecifics. Together, these results suggest life-history advantages of polyploidy or asexual reproduction that exacerbate rather than ameliorate the cost of sex. These outcomes suggest that P availability is unlikely to be a main driver of ploidy polymorphism or the maintenance of sex in P. antipodarum.

Gene model for the ortholog of tango (tgo) in the Sep. 2015 (UC Berkeley ASM127793v1/DbusGB1) Genome Assembly (GenBank Accession: GCA_001277935.1) of Drosophila busckii. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Gene model for the ortholog of raptor in the D. grimshawi May 2011 (Agencourt dgri_caf1/DgriCAF1) Genome Assembly (GenBank Accession: GCA_000005155.1) of Drosophila grimshawi. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

The tribe Spermacoceae (Rubiaceae) comprises a morphologically diverse assemblage of approximately 1,400 species distributed across the Neotropics, Africa, Asia, Australia, and Pacific region. It remains one of the most taxonomically intractable groups in the family, with generic limits repeatedly redefined for more than two centuries. Previous phylogenetic studies based on a limited number of plastid and nuclear markers left numerous relationships unresolved and provided sparse representation of Neotropical lineages. Here, we present the first phylogenomic study of the tribe based on plastome-scale data and expanded sampling of Neotropical taxa. We sampled 121 species representing 55 genera spanning all major clades and generated 123 new plastomes, including 25 species incorporated into a molecular phylogenetic framework for the first time. Maximum-likelihood and Bayesian analyses recovered a highly resolved and strongly supported phylogeny, with uncertainty restricted to a small number of deep backbone nodes. Pollen and seed micromorphology provided additional evidence for evaluating phylogenetic relationships. The resulting phylogenetic framework clarifies generic boundaries across several problematic lineages and supports multiple taxonomic changes. Pervasive homoplasy in seed and floral characters rendered several traditionally recognized genera non-monophyletic, warranting new combinations, including Edrastima oxycoccoides, Stenotis alexanderae, and S. prostrata, and a reassessment of taxa such as Terrellianthus serpyllaceus and Oldenlandia dusenii. We further identify genera requiring additional study and provide an updated key to the 82 recognized genera of Spermacoceae. Together, these results provide the most robust phylogenetic framework yet available for the tribe and establish a foundation for future systematic, biogeographic, and evolutionary research.

Background and AimsThe development of unisexual flowers relies on the tight coordination of flower organ identity and sex determination. The genus Quercus is typically considered strictly monoecious, bearing fully segregated male and female flowers within the same individual tree. However, several reports of atypical flowering across the genus challenge this canonical view, suggesting that flowering in oaks may be more flexible than traditionally assumed. In this work, the dynamics of flower development in Quercus orocantabrica were examined to correlate contrasting floral morphologies with divergent molecular profiles. MethodsThe flowering phenology of Q. orocantabrica trees was closely monitored over several individuals and years, together with a detailed floral morphological analysis of male, female and atypical flowers. Key floral homeotic gene homologues were identified, and their expression assayed in the development of different flowers. Key ResultsRecurrent and widespread hermaphroditic flowering was detected in several Q. orocantabrica trees, frequently associated with unseasonal flowering events. Gene expression analysis of male, female and hermaphroditic flowers revealed a sex-biased expression of Q. orocantabrica B- and C-class genes, with the B-class gene QoPI in particular being tightly associated with the presence of fully-developed stamens. In addition, the expression of the C-class gene QoSHP contrasted with reports in other Fagaceae, highlighting a potential functional divergence of the C/D-class lineage within the family. ConclusionsThe results here depicted indicate that the dynamics of floral sex identity in oaks are more plastic than traditionally assumed, supporting a reinterpretation of oak reproductive biology based on a versatile and resilient framework responsive to different developmental contexts.

Sharks exhibit extraordinary morphological diversity across a wide range of ecological niches, yet large-scale, high-resolution digital datasets of their internal anatomy remain limited. Here we present an open-access 3D shark anatomical repository derived from published X-ray computed tomography (CT) data, featuring manually segmented and systematically annotated models of the chondrocranium, visceral arches, axial skeleton, musculature, and viscera in standard STL format. The dataset comprises 117 individuals, representing 72 species across 25 families and all nine extant shark orders, with 115 full-body reconstructions and two head-only models. This open-access dataset offers a comprehensive resource for comparative anatomy, biomechanical simulations, evolutionary developmental biology and biomimetics research of extant sharks.

In a wide variety of animals, developmental crowding results in adults with smaller bodies. The crowding effect on body size in Drosophila melanogaster is canonically attributed to heightened competition for nutrition. However, whether other consequences of crowding also contribute to its effect on size remains an open question. We tested the relative contributions of nutritional competition, oxygen availability, and larval-generated metabolites to the crowding effect on size. We found that while nutrition explains most of the variation in body size due to crowding, oxygen also contributes in a sex- and nutrition-dependent manner. We found no evidence that larval-generated chemicals affect body size. These data confirm a widely suspected but untested role of nutrition in producing the crowding effect on size in D. melanogaster, while revealing an unexpected role of oxygen, and raise the possibility that behavior may be a mediator of density-dependent plasticity. Research HighlightsWe found that both nutrition and oxygen mediate the crowding effect on size in Drosophila melanogaster.

Coral reefs are constructed by colonial cnidarians whose survival depends on the coordinated growth and physiological integration of thousands of interconnected polyps. While coral skeletons have been extensively studied, the internal three-dimensional organization of coral tissues remains poorly resolved, limiting our understanding of how reef-building corals function as integrated modular organisms. In this study, we established a contrast-enhanced X-ray tomography (XRT) workflow for decalcified coral tissues, enabling detailed visualization and quantitative comparison of internal polyp architecture across four reef-building species with distinct colony forms: Acropora cervicornis, Acropora millepora, Montipora capitata, and Pocillopora damicornis. Importantly, this methodology resolved previously inaccessible patterns of tissue organization and structural connectivity among neighboring polyps. The two Acropora species shared a conserved axial - radial organization but differed in mesenterial morphology, whereas M. capitata exhibited complex, entangled mesenterial networks that connected both neighboring and distant polyps. In contrast, P. damicornis displayed superficial connectivity restricted to the coenosarc. Together, these results suggest that internal tissue architecture is an evolutionarily flexible trait, shaped by ecological and developmental pressures rather than strictly by shared ancestry. Our XRT workflow thus provides a new comparative framework for understanding how corals function as integrated living colonies.