Evolution & Development

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 olfactory system plays a critical role in mammalian environmental perception, with some clades relying on an expanded accessory olfactory (vomeronasal) system (VNS) to detect larger, non-volatile odorants. Mammals make extensive use of this system for social communication between conspecifics. Recent studies have begun to investigate how the VNS changes in response to or as part of ecological transitions. Several studies have identified trends of VNS-associated gene loss or regression in secondarily aquatic mammals. However, continuing discussion on genotype-phenotype correlation within the VNS means that greater effort should be made to investigate the morphology of the VNS in species where it remains poorly understood. Here, we use skeletal and soft-tissue data to demonstrate that the vomeronasal groove, an established osteological correlate for the VNO in bats and primates, is also a valid indicator for its presence in Caniformia. Additionally, we confirm the presence of the VNO in the secondarily aquatic North American river otter (Lontra canadensis) and compare its morphology with that of two close-related species, the semi-aquatic American mink (Neogale vison) and the terrestrial long-tailed weasel (Neogale frenata). This study expands the valid taxonomic scope of the vomeronasal grooves proxy as an osteological correlate, confirms the presence of the VNO in the previously undescribed system of the North American river otter, and highlights the complexity of the mammalian accessory olfactory system.

Arthropods must periodically moult their exoskeleton to permit growth, a conserved developmental process whose morphological and behavioural execution varies widely among lineages. Horseshoe crabs (Limulidae) are members of Xiphosura, a chelicerate lineage with a fossil record extending as far back as the Ordovician and provide a valuable comparative framework for studying the evolution of moulting strategies in Arthropoda. Despite their importance, detailed morphological characterization of moulting in horseshoe crabs remains scarce, limiting developmental studies and broader comparative analyses. Here, we provide a detailed morphological characterization of the moulting process in the Atlantic horseshoe crab Limulus polyphemus. Morphological changes in specific anatomical structures, including the anterior margin of the prosoma, lateral spines and dorsal spinous process of the opisthosoma, were observed during the moulting process. By tracking these morphological markers, such as retraction of the epidermis from the cuticle and degree of corrugation of the epidermis, we were able to identify individuals in the early and late pre-moult stage, predict the onset of ecdysis, and distinguish post-moult and intermoult stages. We compare ecdysis patterns in L. polyphemus with other arthropod taxa, both extant and fossil. We find that, despite differences in behavioural execution, ecdysis in L. polyphemus shares features with other chelicerates, and that both phylogenetic signal and convergent patterns are evident across Arthropoda. This study offers a robust, non-invasive method for determining moult stages in juvenile horseshoe crabs and provides insights into diversity and constraints of ecdysis in Arthropoda.

Nervous systems display extensive diversity in structure and organization, yet a broadly conserved set of signaling pathway components and transcription factors is consistently associated with early neurogenesis in many animal lineages. Determining how these conserved markers map onto the spatiotemporal organization of neurogenic territories across phylogenetically informative but underrepresented lineages, particularly within Spiralia, is critical for refining inferences about the evolutionary origins and diversification of nervous systems. Chaetognaths, a spiralian lineage frequently recovered close to Gnathifera, have a compact and centralized nervous system but lack detailed molecular descriptions of early neural development. Here, we generate an expression-based developmental map of early neurogenesis in the chaetognath Spadella cephaloptera by combining nuclear-staining-based anatomical staging with spatiotemporal analyses of conserved developmental genes associated with early neurogenesis and axial patterning from gastrulation through early post-embryonic stages. Sce-soxB1-like1 and Sce-neuroD expressions mark a lateral neuroectodermal territory during gastrulation. Notably, Sce-neuroD is activated early in a broad ectodermal domain and is expressed within mitotically active neuroectodermal cells, consistent with early deployment in proliferative neurogenic territories. Sce-soxB1 and Sce-soxB2 show delayed and more spatially restricted expression relative to Sce-soxB1-like1, suggesting a paralog-specific partitioning of SoxB deployment during chaetognath neurogenesis. Sce-bmp2/4 and Sce-chd exhibit reciprocal dorsoventral expression during gastrulation that coincides with early neurogenic territory formation, before transitioning to more localized expression later in development. Sce-nk6 and Sce-hb9 reveal early ventral regionalization of the developing ventral nerve center (VNC), with Sce-hb9 occupying a subset of a broader Sce-nk6 domain, in line with conserved ventral subtype-associated regionalization. Sce-th (tyrosine hydroxylase) is detected in a small bilateral subset of hatchling VNC cells, while Sce-dbh (dopamine beta-hydroxylase) is first detected only in early juveniles in the anterior VNC and head domains, suggesting stage-dependent and region-specific deployment of catecholamine-pathway components. Together, these expression-based datasets provide a comparative reference for early neurogenesis in chaetognaths and a framework for assessing conserved and lineage-specific features of early neurogenic patterning across Spiralia.

Skeletal muscle plays important locomotive and metabolic functions, yet its formation and maintenance are processes remaining largely unclear mechanistically in any animal. Teleost fishes display extraordinary muscle growth due to their ability to undergo both hyperplasia and hypertrophy throughout life. These phenomena vary greatly even between closely related species, providing opportunities to elucidate growth dynamics and underlying mechanisms through cross-species comparisons. Using histological and genetic approaches, we compared muscle growth dynamics in three closely related danionin species with distinct growth capacities: the giant danio (Devario malabaricus), the zebrafish (Danio rerio), and Danionella cerebrum, as well as the more distantly related African turquoise killifish (Nothobranchius furzeri). Our study reveals alterations in spatial patterning of muscle hyperplasia and developmental timing to be major contributors to observed differences in muscle growth between examined species. Single-cell RNA profiling, in situ hybridization chain reaction and cell type-specific mutagenesis revealed muscle stem cell-specific expression of extracellular matrix genes that mediate stem cell activity, which in turn may drive growth differences between species. Taken together, our findings highlight autonomous regulation of muscle stem cells as a conserved but adaptable mechanism governing muscle patterning and diversification.

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.

Many animals change their behavior in response to social experiences by learning. Although social learning is adaptive, not all individuals learn. In Heliconius melpomene, H. m. malleti males respond to 2-day prior failed copulation experience by decreasing courtship, whereas H. m. rosina males do not. Here, we explore the transcriptomic differences in both the neural (brain) and sensory (eyes, antennae) tissues underlying this natural diversity in male aversive mate-preference learning. While the transcriptomic profiles of the two subspecies are inherently different across all three tissues, we found the greatest difference between the good (H. m. malleti), and bad (H. m. rosina) mate-preference learners in the brain, followed by the sensory tissues. Known learning genes and Gene Ontology terms were associated with differences in mate-preference learning, suggesting conserved learning pathways across animals. Genes within putative magic loci associated with colors, odors, and locomotion, were also differentially expressed between H. m. malleti and H. m. rosina, suggesting multimodal sensory processing may drive behavioral variance in these two subspecies. Overall, our study identifies genetic underpinnings for differences in preference learning, both in neural processing and sensory tissues. Selection on these genes/networks could result in preference learning-induced reinforcement, leading to reproductive isolation and speciation.

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.

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.

Habitat transitions are a major driver of morphological evolution. Teleost fishes have repeatedly transitioned from benthic to pelagic habitats, often evolving predictable changes in body shape that enhance hydrodynamic efficiency. While freshwater sculpins (Cottidae, Perciformes) are usually benthic, two genera in Lake Baikal, Comephorus and Cottocomephorus, have independently evolved into midwater niches. As sculpins lack a swim bladder, these lineages instead improved buoyancy through reduced skeletal density and increased lipid stores. Using micro-computed tomography and two-dimensional morphometrics, we characterized skeletal evolution across the Baikal sculpin radiation. We found that parallel changes in bone mineral density and microstructure independently evolved in the two pelagic clades. Density reductions occurred throughout the skull in pelagic species. The basibranchials and neurocranium exhibited the lowest overall bone density across all cranial elements. While the jaws maintained the highest absolute density values among the bones we measured, they also showed the greatest proportional reduction in density associated with pelagic habitat use, with a 56.86% decrease in percentage hydroxyapatite and a 21.39% increase in porosity. Morphometric analyses further identified convergence toward an elongate body shape, reduced and posteriorly shifted eyes, and elevated fin insertion in pelagic taxa. These results demonstrate a repeated skeletal lightening and body shape changes accompanying benthic-to-pelagic transitions. This pattern mirrors other benthic-to-pelagic transitions in teleosts that lack swim bladders, highlighting shared biomechanical and microstructural solutions to life in the open water.

The evolution of visual traits in closely-related species living in sympatry is highly influenced by their ecological interactions: while sexual selection tends to promote the divergence of visual cues involved in mate choice, natural selection via predation may promote the convergence of dissuasive signals between prey species, especially in unpalatable or evasive prey. Here, we investigate the impact of sympatry on the evolution of the blue structural colouration in the wings of two closely-related Morpho butterfly species across several localities throughout Central and South America. Dorsal iridescence might affect mate choice and species recognition, which should promote its local divergence among species. However, the bright flashes and dynamic colour patterns produced by iridescence during flight might also increase survival by confusing predators and favouring escape. Such an effect might in turn lead to convergence in wing iridescence between evasive species occurring in sympatry, a phenomenon dubbed evasive mimicry. To test the effect of these putative antagonistic selective forces on visual cues evolution, we quantified the variation of the structural blue colour displayed at 13 different combinations of illumination/observation angles, on the wings of two closely-related Morpho species. We contrasted 10 sympatric and 11 allopatric locations and specifically compared the phenotypic distances between individuals from different species. Phenotypic distances between heterospecific pairs of individuals were significantly smaller in sympatry, consistent with the hypothesis of a local convergence of iridescence due to evasive mimicry. Interestingly, sexual dimorphism was found between males and females, suggesting that the trade-off between natural and sexual selection on the evolution of iridescence might differ between sexes. Our results suggest that local predation pressures may promote repeated evolutionary convergence of structural colouration between evasive prey species living in sympatry.

Echinoderms possess a complex immune system, primarily relying on coelomocytes - immune cells circulating in coelomic fluids. Over the last few decades, various coelomocytes have been described based on morphological features, with holothuroids exhibiting the highest diversity of cell morphotypes among the different echinoderm classes. However, while the overall immune function of these cells is broadly accepted, their respective functions remain unclear, and molecular data specific to the different cell types are still limited in the literature. In this study, we address this gap in functional information and molecular data by using single-cell RNA sequencing (scRNA-seq) on coelomocytes from the perivisceral fluid of Holothuria forskali. We identified 10 distinct clusters, each assumed to correspond to a distinct transcriptional coelomocyte population. Among these, cluster 0 occupies a central position relative to the others, suggesting it may represent "progenitor cells", whereas cluster 6 is markedly divergent from all other clusters. Functional enrichment analyses revealed that some clusters ensure key immune functions, including pathogen recognition, phagocytosis, complement activation and redox balance regulation. In addition, examination of the processed samples under a microscope confirms the presence of a small proportion of recently discovered carotenocytes (7.0%) in the perivisceral fluid, a cell type rich in carotenoids. By using transcriptomics data previously obtained for this cell type by bulk RNA sequencing (bRNA-seq), it was possible to confidently identify cluster 6 as carotenocytes and provide further insights into their gene expression. While further analyses are needed to link other clusters to the different morphotypes previously described in the literature, this pioneer study presents preliminary data on the functional diversity of holothuroid coelomocytes, which could be of broad interest for a better understanding of holothuroid immunity as well as for the study of immune cell lineage evolution across deuterostomes.

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.

Conspicuous coloration in animals is generally thought to evolve and be maintained through inter- or intraspecific interactions such as mate choice, but this might not always be the case. The sight-line hypothesis proposes that conspicuous light-dark contrast in front of the eyes (hereafter, eyeline) evolves and is maintained due to viability selection, enhancing an individual visual acuity and thus evolutionarily associated with a particular foraging behavior that requires accurate aiming. However, empirical evidence that supports the sight-line hypothesis is virtually absent, with no studies demonstrating the key prediction that the direction of eyelines matters. Here, I tested the sight-line hypothesis using macroevolutionary analyses in terns and allies, which are a suitable study system, because they have variation in facial color patterns, including presence/absence and, if any, various angles of eyelines. They also have a large variation in foraging behavior, including picking, plunge diving, and skimming. As predicted by the sight-line hypothesis, tern lineages that require accurate aiming at foraging (e.g., plunge diving) are more likely to have eyelines. In addition, the evolutionary transition to the state with eyelines and these foraging behaviors was more likely to occur than the reverse transition. Furthermore, as expected by the fact that the direction of travel is upwardly deviated from the direction of the bills during skimming, the eyeline angle from bills was evolutionarily positively associated with the occurrence of skimming behavior. To my knowledge, the current study is the first to demonstrate that the direction of the eyeline matters, thereby strongly supporting the sight-line hypothesis.

The brown alga Ectocarpus is a complex yet morphologically simple organism in which cells of the growing filament undergo changes in shape and relative position over time. Here we have investigated the role of cell age, cell position and cell shape in the establishment of cell identity in Ectocarpus. To understand how these factors act and combine to determine cell identity, we used laser capture microdissection (LCM) to isolate specific cell types from young sporophytes of Ectocarpus and then performed differential RNA-Seq analysis. Transcriptome data were used to allocate molecular signatures to cell identities and then cell populations were distinguished on the basis of age, shape, and position. Transcriptome profiling of a wild-type strain provided molecular signatures of five distinct cell identities. To dis-associate cell shape, age and position, we then analysed transcriptomes of two mutants in which the relationships between the three parameters were altered. Collectively our data revealed that molecular cell signatures are dependent primarily on cell position along the filament, and secondarily on cell shape.

Sponges are widely recognized as important model organisms for studying animal evolution, due to their phylogenetic position at the base of the animal tree of life, as well as similarities to the nearest animal relatives, the choanoflagellates. A critical aspect of animal evolution concerns the origin of germ layers, the embryonic structures which give rise to all tissues and organs of animal bodies. Haeckels hypothesis suggested a germ layer homology between sponges and corals, and thus all eumetazoans (complex animals including cnidarians and bilaterians). According to this hypothesis, sponge choanoderm (composed of the feeding cells, choanocytes) and sponge pinacoderm (the outer epithelium) would be homologous to eumetazoan endoderm (from which the digestive system originates) and the ectoderm (giving rise to the epidermis), respectively. We addressed this hypothesis comparing tissue-specific transcriptomes derived from single-cell transcriptome datasets of sponges and cnidarians. We have sequenced single cell transcriptomes of Australian calcareous sponge, Sycon capricorn, and identified its cell types using a combination of in silico annotation of the cell clusters and in situ hybridization with marker genes. Single-cell transcriptome datasets for two demosponge species and two cnidarian species were extracted from recent literature. Homology was assessed using the SAMap algorithm, which has been designed to identify homologous cell types across vast evolutionary distances by detection of shared expression profiles. Our results are fully consistent with Haeckels hypothesis, supporting homology between the innermost layers of sponges and cnidarians (choanoderm and endoderm/gastrodermis) as well as the outermost layers of sponges and cnidarians (pinacoderm and ectoderm/epidermis). Thus, sponge body plan appears to represent an intermediate step between single cell protists (choanoflagellates) and complex animals, rather than being independent experiment in animal multicellularity as suggested by alternative hypotheses.

In birds, the quadrate bone serves as a hinge articulating with the lower jaw and the skull, playing an important mechanical role in the feeding apparatus. Avian cranial kinesis is dependent on the streptostylic quadrate transferring force from the adductor muscles at the back of the skull toward the beak, as part of a four-bar mechanical linkage to elevate and depress the bill. The complex morphology of the bird quadrate has led to authors adopting a range of alternative terminologies to describe the same anatomical structures and character states, impeding clarity of communication and presenting a barrier to progress in our understanding of the evolution of this important component of the avian feeding apparatus. Here, we reconcile terminological discord among previous studies on avian quadrate morphology and propose a stable nomenclature for future work. To characterise the considerable variation in quadrate form across crown bird diversity, we present an extensive anatomical atlas of the avian quadrate and summarise major patterns of quadrate morphological variation across extant avian phylogeny. In addition, we investigate macroevolutionary patterns in avian quadrate morphology, incorporating comparisons of crown birds and Late Cretaceous near-crown stem birds. We demonstrate that quadrate characters are useful for diagnosing a range of major avian subclades, and suggest that numerous distinctive features are likely to be associated with important biomechanical consequences. This investigation has implications for resolving the unsettled phylogenetic relationships of extinct bird clades such as Pelagornithidae and Gastornithiformes, as well as controversial relationships within several extant groups.

How evolutionary and developmental processes interact to determine axes of neural variation that produce behavioural diversity has been debated for many decades, with alternative hypotheses giving differential emphasis to functional coupling, which favours co-evolution, and developmental constraint, which enforces it. A critical omission is data on the genetic architecture of brain size and structure, which more closely illuminates the shared developmental dependencies between components of an integrated system. Here, we exploit ecological divergence between Astatotilapia calliptera and Aulonocara stuartgranti, two closely related cichlid species from Lake Malawi, to explore the genetic architecture of brain evolution. Using computer vision and machine learning techniques to extract volumetric data from micro-tomographic images, we first demonstrate significant divergence in brain composition between these species. Genomic and micro-tomographic imaging data from a population of hybrids generated between the two species were used to investigate genetic factors shaping this differentiation. We show that the majority of brain components are integrated phenotypically in hybrids, but genetic correlations between them are generally weaker. We further show that variation in multiple brain components is associated with variation in largely structure-specific quantitative trait loci, rather than determined by genetic factors with broad effects across the entire brain. These results suggest a genetic architecture that can facilitate modular changes in brain structure, and imply that individual components are independently evolvable.