Evolution & Development

Understanding how anatomical structures evolve requires disentangling the roles of integration and modularity in shaping morphological variation. The vertebral column, a serially repeated and regionally differentiated structure, provides a powerful system for investigating these processes. Here, we examine how vertebral morphology evolves in relation to whole-body elongation across the adaptive radiation of Lake Malawi cichlid fishes. We tested for evolutionary integration between the precaudal and caudal domains, as well as assessed the contributions of vertebral count, centrum shape, and intervertebral spacing on body elongation. We find strong evolutionary integration between precaudal and caudal vertebral shape, with both vertebral shapes varying along shared axes of multivariate shape change. Despite this, precaudal and caudal vertebral counts evolve independently, indicating a decoupling between the evolution of identity and morphology. Whole-body elongation is significantly associated with coordinated changes in vertebral and rib morphology, including proportional increases in centrum size, posterior displacement of neural and haemal spines, and increased rib curvature. In contrast, centrum elongation and intervertebral spacing do not independently explain body elongation beyond vertebral counts. These results demonstrate that body elongation in cichlids necessitates integrated, multivariate changes in axial morphology. Our findings highlight the importance of morphological integration in facilitating coordinated evolutionary responses in anatomical systems.

The diversity of body shapes is one of the most prominent features of phenotypic variation in mammals. Yet, mammalian body shapes are poorly quantified and the underlying components contributing to its diversity as well as its relationship to other components of the skeleton are rarely tested. Here, we use lagomorphs (hares, rabbits and pikas) as a model system to (1) investigate which components of the skeleton contributed the most to body shape diversity, (2) examine the relationships between body shape and relative limb lengths, and (3) test how body size, ecotype, burrowing behavior, and locomotor mode influenced variation in lagomorph body shape and appendicular morphology. We quantified the body shape and functional proxies of the appendicular skeleton in 40 lagomorph species from osteological specimens held at museum collections. Using phylogenetic comparative methods, we found the relative length of the ribs and elongation or shortening of the thoracic and lumbar regions contributed the most to body shape evolution across lagomorphs. Second, we found that only leporids (hares and rabbits) exhibited a significant relationship between limb length and body shape, where more elongate species exhibit relatively shorter forelimbs and hindlimbs. Lastly, we found that models incorporating body size were the best predictors of lagomorph body shape and the majority of the appendicular traits, whereas models incorporating burrowing behavior and locomotor mode were largely poor fits. Broadly, these results indicate that larger lagomorphs tend to exhibit more robust body shapes with longer, more gracile forelimbs, whereas smaller lagomorphs tend to exhibit more elongate body shapes with shorter, more robust forelimbs. Overall, this work contributes to the growing understanding of mammalian body shape evolution and demonstrates the importance of not omitting body size in ecomorphological analyses.

In sex-role reversed species, females are socially polyandrous and compete for multiple mates, whereas males conduct the majority of parental care. To understand the extent to which physiological differences between females and males are shaped by sex roles, we examined sex differences in gene expression in sex-role reversed northern jacanas (Jacana spinosa). Given that females compete for mating opportunities, and males cycle between courtship and parental care, we predicted that transcriptomic profiles would be more similar between females and courting males, in contrast to female and parenting males. Leveraging a high quality de novo genome assembly, we conducted RNA-seq on two brain regions associated with the regulation of social behavior: the preoptic area of the hypothalamus and the nucleus taeniae. The majority of genes differentially expressed between the sexes were male-biased. Of these male-biased genes, the majority were located on the Z-chromosome. Contrary to our prediction, the greatest difference in autosomal gene expression was between females and courting males, in the preoptic area of the hypothalamus. Several differentially expressed genes related to elements of hormone signaling that are likely to be behaviorally salient, including higher expression of androgen receptor in females relative to parenting males, and higher expression of prolactin receptor in males, regardless of breeding stage. Some sex-associated gene networks were also associated with competitive traits, whereas others were associated with aggressive behaviors, regardless of sex. Few genes were differentially expressed between courting and parenting males, yet some nonetheless had connections to behavioral endocrinology, including prolactin, thyroid and insulin-like growth factor pathways. Our investigation of sex differences in gene expression can help to reveal the molecular mechanisms underlying female competition and male parental care in socially polyandrous species. We conclude that social polyandry is not a simple reversal in the direction of sex-biased gene expression in the brain, but rather a result of complex genetic and hormonal interactions that warrants further study.

The evolution of body shape reflects the interplay between functional constraints and habitat structure. In fishes, cave environments are well known for promoting regressive traits such as eye and pigment loss, yet their influence on overall body form remains poorly understood. Here, we examine patterns of body shape variation in cave- and surface-dwelling trichomycterid catfishes from northeastern Colombia to assess whether consistent associations exist between habitat type and morphology. Using geometric morphometric analyses, we quantified differences in body shape among species inhabiting subterranean and surface environments. Our results reveal significant habitat-associated differentiation in body shape along the main axes of morphological variation. Cave-dwelling species exhibit more elongated and fusiform body shapes, whereas surface-dwelling species tend to show deeper and more robust morphologies. In a functional context, these contrasting body patterns suggest associations with differing locomotor demands imposed by subterranean versus surface habitats. Although we do not explicitly test convergence or performance, the recurrence of similar body shapes among species from different clades occupying comparable habitats is consistent with repeated morphological responses to shared ecological constraints. Research HighligthsO_LIMultivariate shape analyses reveal significant habitat-associated variation in trichomycterid fishes. Recurrent morphological patterns suggest repeated responses potentially mediated by habitat constraints. C_LIO_LIBody shape differs consistently between cave- and surface-dwelling trichomycterids. Cave species exhibit more elongated and fusiform forms, whereas surface species display deeper body configurations. C_LI

The presence of multiple discrete color patterns within a species has captivated evolutionary biologists for more than a century, especially when such polymorphism is confined to one sex. The brown anole Anolis sagrei exhibits a female-limited polymorphism in dorsal patterning, which is controlled by allelic variation at the autosomal gene CCDC170. Here, we present and test a threshold model that can explain why the polymorphism is female-limited. We hypothesize that allelic variation at the CCDC170 locus affects only female color pattern because this gene is co-expressed with its neighboring gene ESR1, highly expressed in female, but not male, embryos. By manipulating embryonic estradiol levels, we show that genetic males can be induced to express the polymorphism according to allelic variation at the CCDC170 locus, which is naturally masked by low expression levels of this gene. Inversely, treating genetic females with fadrozole, which depletes estradiol, leads to monomorphic patterns irrespective of genotype, as for natural males. Using RT-qPCR, we demonstrate that these effects are accompanied by a direct influence of estradiol and fadrozole on gene expression levels of CCDC170 and ESR1, thereby validating the threshold model. Our results suggest that the CCDC170-ESR1-locus is part of a mechanistic link between the morph-determining and the sex differentiation systems and provide a causal explanation for the developmental origin of a sex-limited color polymorphism.

The morphogenesis of complex vertebrate appendages requires precise regulation of growth, governed by distinct positional identities. The zebrafish caudal fin achieves a symmetrical, forked morphology through the regional specialization of the bony rays: peripheral rays are composed of relatively long, thick segments; while the central rays are made up of shorter, thinner segments, and their overall length is restricted. This length differential establishes the definitive forked shape of the organ. We asked whether these regional morphological differences reflect distinct underlying positional identities. Transcriptomic profiling of intact tissues from adult wild-type zebrafish suggested that central rays possess unique expression profiles, distinct from those of peripheral rays. We previously identified a treatment during embryogenesis that allows excess growth in the central rays, creating a truncate fin shape in adults-we asked whether this novel fin shape was caused by a peripheralization of the central rays. Indeed, the central rays of truncate fins were not only longer, but were composed of longer and thicker individual segments, reminiscent of peripheral rays. Further, gene expression in the central regions of truncate backgrounds showed signatures of peripheral identity. During development of the truncate phenotype, peripheral markers became expressed in more central domains of the growing truncate caudal fin, and in the supportive endoskeleton, the central hypural diastema was lost from the earliest stages. Ultimately, our results demonstrate how adult morphologies may be altered by shifts in positional identities. These findings clarify the anatomical patterning and molecular profiles that underlie regional specialization during caudal fin development.

Factors varying along elevational gradients impose strong aerodynamic and physiological constraints on powered flight, yet the internal anatomical correlates of flight performance in animals under such conditions remain poorly understood. In hummingbirds, sustained hovering requires extreme muscular power output, making the pectoralis muscle a key interface between environmental constraint and performance. We tested whether elevation is associated with variation in pectoralis microanatomy across three hummingbird assemblages spanning a [~]1500 m gradient in the Colombian Andes. Using tissue morphometry of trichrome-stained transverse sections of the pectoralis, we measured interstitial collagen fraction as a proxy for extracellular matrix investment and quantified fiber cross-sectional area, packing density, and size heterogeneity. Collagen investment varied across elevational bands, peaking at mid elevation ([~]1750 m) and declining toward high elevation ([~]2600 m). In contrast, muscle fibers were smaller and more densely packed at higher elevations. Variation among species was small relative to differences among elevational assemblages. Formal model comparisons provided limited support for non-linear responses to elevation, indicating that patterns across traits are better explained by interacting constraints than by a single monotonic response to factors varying along elevational gradients. These results show that hummingbird flight muscle microanatomy varies with elevation in a trait-specific manner, with the strongest evidence in fiber geometry. More broadly, our findings highlight that multiple components of muscle microarchitecture, including the extracellular matrix, vary in a context-dependent manner across elevational gradients in an extreme volant system.

In tunicates, larvaceans represent a fascinating case of evolution, where the chordate body plan has been maintained despite a rapidly evolving genome characterized by strong In contrast to other tunicates, larvaceans keep the chordate body plan during their entire life. They have acquired a highly specialized epithelium in charge of producing the "house", a complex extracellular apparatus used for filter feeding in the plankton. To what extent the house and this epithelium represent true molecular innovations withing chordates is a question for which thorough transcriptomics can bring novel insights. We conducted a developmental profiling of gene expression at the single-cell level in the larvacean Oikopleura dioica. We provide detailed descriptions of cellular transcriptomes associated with the house-synthesizing organ, which permits to define the molecular specifics of epithelial cell territories. We followed their emergence during development, and we identified genes that represent key candidate molecules for regulating the morphogenesis of the house-producing organ. Dynamic changes in gene expression and cell identities during major developmental transitions of the lifecycle illustrate that our dataset effectively allows access to the diversity of O. dioicas cell types in embryos and in adults. The resources presented here constitute critical assets to investigate larvacean biology and evolution for mechanistic and comparative goals.

The evolution of visual systems has compelled numerous investigations of developmental processes underlying eye patterning across Bilateria. It is well-established that homologs of the transcription factor Pax6 play a highly conserved role in eye fate specification and are at the top of the retinal determination gene network (RDGN) hierarchy. In insects, the two Pax6 homologs eyeless (ey) and twin of eyeless (toy) are required for the development of the two visual systems broadly found within the phylum (i.e., median and lateral eyes). Curiously, Pax6 homologs do not appear to maintain this function in well-studied chelicerate models, with emphasis on spiders, a lineage of arachnids with great diversity of eye form and acuity. It was recently proposed that the gene Pax2 (shaven; sv) may have subsumed the role of eye fate specification in chelicerates, a hypothesis predicated upon the observation that one of two spider Pax2 copies is strongly expressed in the developing lateral eyes during embryogenesis. However, no functional data are available for any Pax homologs across Chelicerata. We examined the incidence of Pax family genes across Chelicerata, as well as interrogated the expression and function of Pax2 and Pax6 homologs in the daddy-longlegs Phalangium opilio, an arachnid recently discovered to bear a highly plesiomorphic arrangement of visual systems. Here, we show that ey and toy are expressed early in the developing head lobes of P. opilio, whereas sv is not expressed until well after stages when downstream RDGN members (eyes absent and sine oculis) are already activated. Gene silencing of ey, toy, and sv individually had no discernible effect on eye development. By contrast, double knockdown of ey and toy resulted in an array of median eye defects, spanning loss of some cells of the eye to total loss of the median eyes. Gene expression assays also showed that depletion of the two Pax6 copies resulted in failure of the vestigial median and vestigial lateral eyes. These data are consistent with a conserved role for Pax6 homologs in patterning both visual systems and all three eye pairs in the daddy-longlegs. Our results comprise the first functional data for Pax6 genes in any chelicerate and suggest that heterochronic shifts in expression, rather than changes in function, underlie the atypical dynamics of Pax genes in derived arachnid groups such as spiders.

The head of chelicerates, such as spiders, scorpions and mites, is composed of the ocular, chelicerae and pedipalp segments and is considered to be homologous to the procephalon of insects which comprises the ocular, antennal and intercalary segments. Head segmentation in the spider, Parasteatoda tepidariorum, is a dynamic process in which a single stripe of expression of the P. tepidariorum hedgehog (hh) gene splits twice to form three separate stripes, which help pattern the three spider head segments. This dynamic hh stripe splitting process is dependent on spider homologues of the transcription factors orthodenticle (otd) and odd-paired (opa). Here we investigate the conservation of this dynamic patterning mechanism in two insect models: the hemimetabolous pea aphid, Acyrthosiphon pisum, and the holometabolous red flour beetle, Tribolium castaneum.. We show that insect hh, otd and opa homologues are expressed in a highly conserved temporal and spatial pattern during procephalon development in these insects. Our data are consistent with an ancestral insect state in which a single hh stripe splitting event underpins patterning of the ocular and antennal segments, followed by de novo formation of the intercalary hh stripe. Using parental RNAi in T. castaneum, we show that hh, otd and opa homologues exhibit striking similarities in their regulatory interactions during spider and insect head/procephalon segmentation. Our data suggest that hh, otd and opa homologues contribute to an ancient and largely conserved gene network controlling head/procephalon patterning in arthropods. We discuss the implications of these data for our understanding of the origin and evolution of the arthropod head, and propose a new model for the evolution of anterior patterning in holometabolous insects.

Although cartilage in tetrapod skeletons is typically said to lack blood vessels, this is only true for adult cartilage. In young bird and mammal cartilage, a dense network of vasculature-containing tunnels --cartilage canals-- perforate the growing skeleton, helping nourish the cartilage and develop the ossification centers that will later form the skeletons epiphyseal bone. As the canals and their rich vascular network typically recede as animals age, the healthy cartilage of adult animals is typically known to be avascular. Here, however, we use a range of tissue characterization and visualization techniques --including light/electron microscopy and microCT-- to show that the skeletons of rays and sharks (elasmobranch fishes) not only possess cartilage canals, but that these structures persist in the adult skeleton. The morphology and tissue composition of elasmobranch cartilage canals argues homology with mammalian cartilage canals and an ancient invasion of the vascular system into cartilage. However, the anatomical location of canals --extending away from mineralized tissue not toward it-- and the lack of endochondral ossification in ray and shark cartilage suggest that cartilage canals developed early in vertebrates as a transport system for nutrients and mesenchymal cells into the growing skeleton. We describe distinctive features and variation in elasmobranch cartilage canals, discuss their possible roles and their potential for tissue mineralization, and the biomedical implications for their presence in a clade of animals with continuously growing cartilaginous skeletons.

Medusozoan cnidarians (e.g., jellyfish) metamorphose from a benthic juvenile polyp into a pelagic adult medusa, providing a well-known example of a clade that uses tissue remodeling to create distinct juvenile and adult body plans. Staurozoans (i.e., stalked jellyfish) are an atypical lineage of medusozoans that have lost their medusa stage; thus, their juvenile and adult body plans look remarkably alike. Their limited metamorphosis is characterized by the regression of primary (juvenile) tentacles and the development of secondary (adult) tentacles. In some staurozoan lineages, metamorphosis also involves development of novel adhesive structures (anchors), which are built on top of the regressing primary tentacles. Understanding how cells are partitioned from making juvenile tissues to making adult tissues is important for understanding how animals can make adult structures in the absence of complete metamorphosis. We compared the abundance and distribution of proliferative cells in tissues undergoing regression (primary tentacles) and development (secondary tentacles and anchors) during the juvenile to adult transition in the San Juan Island stalked jellyfish, Haliclystus sanjuanensis. We show that proliferative cells are lost in regressing primary tentacles but are gained in anchors, consistent with a shift in investment from juvenile to adult tissue. Prior to regression, primary and secondary tentacles show similar patterns in their proliferative cell distribution and in the identity of their cnidocytes (stinging cells), indicating that adult tentacles are made by re-deploying a juvenile tentacle program. Finally, we demonstrate that unlike secondary tentacles, primary tentacles cannot regenerate, illustrating that the temporary investment in this tissue is tied to their loss of proliferative cells. Thus, we propose that continued investment in a population of proliferating cells is an important mechanism for segregating temporary tissues (primary tentacles) from long-term tissues (secondary tentacles). These observations of cell dynamics in H. sanjuanensis suggest that temporary investment into juvenile structures may be used to pattern novel adult tissues, providing an important mechanism for diversifying adult body plans.

Metridium senile is a clonal anemone that engages in fighting interactions to defend its territory from non-clonemates utilizing an inducible fighting organ, the catch tentacle. Upon contact with a non-clonemate, the catch tentacle tip detaches onto the non-clonal individual, resulting in necrosis where the tip attaches to the non-clone. The incapacitating function of the catch tentacle is driven by a unique type of cnidocyte, the holotrich, which is not found elsewhere in M. senile, including the feeding tentacles from which catch tentacles develop. Metridium farcimen, the sister species to M. senile, never develops catch tentacles despite their close phylogenetic relationship, as exemplified by their ability to hybridize. Here, we compare the feeding tentacles of both species to the catch tentacles in M. senile to determine how catch tentacles achieve their unusual function. We found that the feeding tentacles of M. senile and M. farcimen house similar types of cnidocytes that develop from proliferative cells distributed throughout the tentacle. By contrast, catch tentacles house distinct cnidocyte types from feeding tentacles and restrict proliferative cells to the base of the tentacle. This suggests immature cnidocytes migrate from base to tip to replace lost cells after an aggressive interaction in the catch tentacle. Additionally, we observed two morphologically and chemically distinct types of holotrichs in the catch tentacles that appear to use different cues to induce firing. Together, our data suggests that the novelty of catch tentacle aggression is mediated by distinct cell and tissue dynamics.

Dormancy is a widespread adaptive strategy that allows organisms to survive in temporally varying habitats by suspending development and reproduction. Although environmental variability is expected to shape dormancy strategies, it remains unclear how differences in environmental variability and predictability influence both the production of dormant embryos and the termination of dormancy. We addressed these questions by comparing D. pulex and D. obtusa, two closely related species that inhabit environments differing in variability and predictability. We hypothesized that D. obtusa, which inhabits ephemeral environments, would exhibit a greater propensity for sexual reproduction and dormancy and would require stronger cues to break dormancy than D. pulex, which occurs in more permanent, predictable habitats. Consistent with our hypothesis, D. obtusa lineages produced significantly more males and ephippia than D. pulex when reared under identical laboratory conditions, indicating greater investment in sexual reproduction and dormancy. Contrary to our hypothesis, we found no difference in responsiveness to cues between the two species. Across species, embryos broke dormancy and hatched most readily after experiencing changes in cold and light, even if not experienced at the same time. In contrast, desiccation reduced the propensity to break dormancy. Together, these results indicate that species occupying more ephemeral environments invest more heavily in the production of dormant offspring, but that the environmental cues regulating dormancy termination appear broadly similar between species. This pattern suggests that while investment in dormancy may evolve in response to environmental variability, the mechanisms controlling dormancy termination are more conserved.

AO_SCPLOWBSTRACTC_SCPLOWEnvironmental heterogeneity across freshwater systems often promotes phenotypic variation, yet disentangling environmentally induced variation from heritable differentiation remains a central goal in evolutionary ecology. We investigated the geographic distribution and morphological differentiation, and heritability of shell traits among populations of the freshwater lymnaeid snail Pectinidens diaphanus in Patagonia. Extensive field surveys across 196 freshwater sites revealed that the species occupies a broad range of lentic and lotic habitats and constitutes the only lymnaeid inhabiting southern Patagonia. While reproductive anatomical structures were conserved across populations, shell shape differed markedly among populations from contrasting habitat types, with population identity explaining nearly 50% of total shape variation. Populations from hydrologically unstable habitats (ponds and streams) exhibited more elongated shells and relatively smaller apertures, a pattern consistent with functional responses to hydroperiod variability and desiccation risk. To assess the heritability of this differentiation, we conducted a common-garden experiment across two generations. Shell shape differences between permanent- (lagoon) and temporary- (pond) habitat-derived populations persisted into the G2 generation reared under standardized laboratory conditions, indicating that the observed variation is not solely a response to local environmental conditions but includes a heritable component. Together, our findings demonstrate that P. diaphanus constitutes the sole lymnaeid across southern Patagonia, occupying a broader range than previously documented, and that populations show heritable shell differentiation potentially associated with contrasting freshwater habitats. By integrating large-scale biogeographic surveys with morphometric and experimental approaches, this study provides new insight into how habitat variation may contribute to ecological and evolutionary differentiation in freshwater gastropods.

Sarita Lake, British Columbia houses a distinctive population of threespine stickleback (Gastrosteus aculeatus L.) with a phenotype characterized by unusually large individuals relative to nearby conspecifics. We tested the hypothesis that members of this population are not isometrically larger but rather exhibit variation in allometric trajectories that reflect changes in developmental timing impacting the developmental-genetic architecture of the phenotype. We used 3D geometric morphometrics to characterize the size and shape of skulls, pectoral girdles and pelvic girdles from a sample of individuals from nearby freshwater and marine populations and compare them to a sample from Sarita Lake. We showed that individuals from the Sarita Lake population are larger in each body region compared to most other populations examined. Further, these individuals have dorsally expanded skulls and relatively robust pelvic armour. We also showed that the relationship between size and shape is differently structured among body regions and is heavily influenced by non-uniform sexually-mediated variation across populations sampled. Our results reflect complex underlying developmental trajectories, and we suggest that the large phenotype observed may be driven by fecundity selection on female size in combination with a limnetic trophic niche and relatively increased predation pressure in Sarita Lake.

Silk glands have been found in two groups of amphipods: the Corophiida and the Ampeliscidae. The silk glands in Ampeliscidae, however, have yet to be examined in detail. Here we report, for the first time, the morphology and distribution of pereopodal glands in the Ampeliscidae, in non-thread producing Synopiidae, and in the Paragammaropsidae. In the Ampeliscidae we found two gland types distributed throughout all pereopods which have the ability to create threads. Pereopods three and four have additional silk extrusion morphology at the tip of the dactylus in which silk is transformed into semi-cylindrical threads used for building domiciles. Synopiid outgroup species have one of the gland types but lack silk extrusion morphology. Using ancestral state reconstruction analysis, we find that glands in the Synopiidae are likely ancestral and hypothesize that silk glands in Ampeliscidae are derived from these ancestral glands. Silk-spinning pereopods in the Paragammaropsidae had similarities with both Corophiida and Ampeliscidae but had distinctions. Ampeliscidae silk-spinning systems bear surprising resemblance to the Corophiida which presents one to reconsider the taxonomic placement of Ampeliscidae and the origins of silk-spinning in amphipods. This is the first comprehensive study on the glandular systems of Ampeliscidae, Synopiidae, and Paragammaropsidae using advanced microscopy, providing pertinent morphological data to the study of arthropod silk gland evolution and complex traits.

Life history strategies such as rapid growth and high population turnover rates observed in vertebrates have been thought to have emerged relatively late in evolution. However, very little direct evidence exists at the species level for early vertebrates. In this study, a large fossil collection of over 450 specimens of Protaspis spp, heterostracan agnathans from the Cottonwood Canyon Formation at Beartooth Butte, Wyoming, from the Early Devonian, was analyzed. Morphological observations, analysis of bone plate completeness, and length-frequency analyses using ELEFAN--commonly used in recent fish studies--were applied to reconstruct growth rates, cohort structure, and ontogenetic processes. Protaspis specimens exhibited a continuous growth series from juvenile to adult stages, and a clear cohort structure was identified from the length-frequency distributions. The ELEFAN analysis suggested a life-history characterized by rapid growth and a short life span, and these features remained consistent in subset analyses restricted by species or locality, confirming the robustness of the estimates. Furthermore, the integration of the dermal bone plates progressed during the late stages of ontogeny, revealing that the rapid growth during the juvenile stage preceded the completion of this defensive structure. Comparisons of their growth parameters with those of extant fishes show that Protaspis does not align with slow-growing, long-lived "living fossil" taxa, but instead clusters with small-bodied, fast-growing species. These findings suggest that life-history strategies involving rapid growth and high population turnover were already established in early jawless vertebrates, much earlier than previously assumed.

Previous genetic studies of skeletal variation in threespine stickleback fish (Gasterosteus aculeatus) have focused primarily on striking morphological differences. Here, we examine the largely unexplored genetic architecture of internal bone microstructural variation between marine and freshwater stickleback. {micro}CT X-ray analysis revealed differences in the porosity, bone thickness, and bone volume fraction within armor plates and vertebrae from a marine and freshwater stickleback. Quantitative trait locus mapping in F2 progeny from a marine x freshwater stickleback cross identified a significant locus on chromosome 4 influencing multiple aspects of armor plate internal microstructure. This locus overlaps the well-characterized Eda region previously known to control armor plate number and size. Co-mapping of bone microstructure could be due to pleiotropic effects of Eda on multiple aspects of plate development or to changes in closely linked genes including Itm2a, which also plays a role in bone formation. Most bone microstructure traits in vertebrae showed weak or no genetic signal, consistent with a polygenic architecture. However, we identified a highly significant locus on chromosome 17 that is strongly associated with abnormally thickened "ivory vertebrae" that occurred in 8.4% of F2 offspring. This phenotype resembles Pagets disease in humans, and the major locus region contains Tnfrsf1b, the stickleback ortholog of a human Pagets disease susceptibility gene TNFRSF11A. Together, our findings identify genetic loci underlying natural variation in bone microstructure in wild fish and reveal a candidate gene associated with a disease-like skeletal phenotype, highlighting stickleback as a model for studying both evolutionary and pathological bone biology.

Following the evolution of internal fertilisation, the female reproductive tract became the site of reproductive interactions. However, our understanding of the evolution of female reproductive tract function, including postmating responses critical for reproductive success, are taxonomically limited. Traumatic insemination in the common bedbug (Cimex lectularius) presents an unusual scenario under which postmating responses unfold. Bedbugs have evolved a novel organ, the mesospermalege, that is the site of initial ejaculate x female interactions. As the female reproductive tract does not take receipt of the ejaculate until several hours after mating, bedbugs provide a unique opportunity to explore the evolution of a novel reproductive organ that decouples postmating female responses involved in mating and transfer of the ejaculate from sperm storage, ovulation, and oviposition. Here we show that the mesospermalege has a gene expression profile consistent with functions of ejaculate processing and immune response normally found in the lower reproductive tract of other insect species. In parallel, the postmating response in the lower female reproductive tract is delayed, coinciding with movement of sperm through the female, clearly showing that the postmating response has evolved in response to sperm receipt rather than being an innate function of the tissue. Notably, we also found expression of male seminal fluid genes in the mesospermalege, indicating that intersexual molecular dynamics influence the evolution of reproductive tissues. Our results provide insights into the evolution of novel reproductive traits and female postmating physiology in a global pest with an unusual reproductive biology. SIGNIFICANCEReproduction poses one of the most persistent challenges faced by animals whereby females undergo a series of physiological changes after mating. The independent origin of a reproductive organ in bedbugs (called the mesospermalege) which has evolved to alleviate the costs of traumatic insemination presents a unique case to study the evolution of a novel trait and postmating physiology. Using transcriptomics, we show that many genes normally expressed in the female reproductive tract are instead expressed in the mesospermalege. The reproductive tract also shows a delayed postmating transcriptional response coinciding with sperm entry into the reproductive tract. Our results provide insights into the evolution of reproductive traits and female postmating physiology in a global pest with an unusual reproductive biology.