EvoDevo

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.

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.

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.

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.

Adult hydrozoan cnidarians undergo extensive tissue turnover, generating neural cell types including nematocytes (stinging cells) and gland cells from interstitial stem cells (i-cells) expressing stemness proteins such as Piwi and Nanos. The contribution of i-cells during embryogenesis, however, has been unclear. Here we address the origin of neural cells during development of the Clytia hemisphaerica planula larva. Marker gene in situ hybridisation revealed that Piwi/Nanos1-expressing cells within the early gastrula presumptive endoderm generate a substantial pool of nematoblasts, a few of which migrate and differentiate in the planula ectoderm. Some neurogenic and neuronal markers, however, showed a markedly distinct expression profile, developing within a basal layer of the aboral/lateral ectoderm during gastrulation. Embryo bisection and lineage tracing experiments confirmed that sensory neurons and secretory cell types derive from gastrula ectoderm, while nematocytes and at least some ganglionic neurons derive from i-cells. Knockdown and inhibitor treatments revealed steps in neuron and nematocyte development regulated by Wnt-{beta}-catenin. We conclude that two distinct neurogenesis pathways operate during Clytia embryogenesis, one involving aboral ectoderm delamination, and one generating mainly nematocytes from i-cell-like precursors. Summary statementDuring embryogenesis in the hydrozoan Clytia neural cell types derive both from Piwi/Nanos expressing "i-cells" and from ectodermal delamination during gastrulation.

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.

Jellyfish have complex life-cycles, but there has been limited exploration of how this is achieved at the cellular level. We used single-cell transcriptomics to assemble a cell atlas for the planula larva of Clytia hemisphaerica, and compared it to an updated cell atlas for the medusa (jellyfish) stage. The cells of the planula fell into the same broad categories as for the medusa: ectoderm, gastroderm, interstitial cells (i-cells), nematocytes (stinging cells), neurons and secretory cells. Although the planula cells generally showed less diversity than medusae within each category, cells with specialized features unique to their stage could be distinguished by their transcriptional profiles as well as by ultrastructure. Some planula-specific types were identified: aboral secretory cells involved in settlement, and a cell type attributed a role in immunity or post-metamorphic theca production. Distinct transcriptome profiles within different regions of the ciliated planula ectoderm reflected different post-metamorphosis fates of domains along the oral-aboral axis. Inspection of the cell clusters showing significant similarity of marker genes between planula and medusa, and inference of similarity using a statistical model of marker gene presence/absence, revealed correspondences between families of cells from planula and medusa rather than precise cell identities.

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.

Animals sense and integrate complex external cues to make developmental decisions that help them better survive and adapt to their natural habitats. Under environmental adversity, nematodes can enter an alternative developmental pathway to form a diapautic and stress-resistant stage, termed the dauer larvae. While dauer formation has been well characterized in Caenorhabditis elegans, how environmental factors influence analogous stages in other nematode species remains largely unexplored. This study examines how symbiotic bacteria, temperature, and pheromones affect the formation of the infective juvenile (IJ), a dauer-like stage, of the insect-parasitic nematode Steinernema hermaphroditum. In contrast to C. elegans, where dauer entry is promoted by heat, IJ development in S. hermaphroditum development is enhanced by reduced temperature. Moreover, the presence and absence of live symbiotic bacterium Xenorhabdus griffiniae functions as an ON-and-OFF switch that regulates the host IJ formation. Crude pheromone extracts from S. hermaphroditum liquid culture do not robustly induce IJ formation in a dose-responsive manner, unlike the potent pheromone-driven dauer entry observed in C. elegans. Nutrient-rich liver-kidney media that mimics host insect environment showed IJ entry induction in a pheromone-dependent manner. These data suggest that external cues, such as temperature, microbial diet, and pheromone, are perceived differently by S. hermaphroditum in comparison to that of C. elegans, reflecting species-specific adaptations to distinct ecological niches and life history strategies.

To achieve a stereotypic lineage, each embryo of Caenorhabditis elegans follows an invariant cell differentiation process arising from a combination of cell polarisation, asymmetric or symmetric divisions, combined with intercellular signalling processes. This pattern of embryonic cell differentiation is driven by regulated segregation of molecules occurring at each cell division, including polarity proteins or cell fate determinants, transcription factors, p-granules and mRNAs. These distribution patterns are coupled with a robust spatio-temporal orchestration of cortical actin dynamics, which also plays a crucial role in these processes. However, compared to other molecular contents, how the actin per se is segregated from the first asymmetric division onward remains poorly understood. This study presents a thorough quantification of the intracellular distribution from the zygote to the 4-cell stage of key actors related to actin polymerisation: two nucleators (a formin and the Arp2/3 complex), a capping protein and E-cadherin. We additionally developed a novel method to assess actin polymerisation capacities from single blastomere extracts. We found that actin-related signatures arise at these early stages and that differential mechanisms of protein segregation and homeostasis occur, depending both on the cell pair and on the protein considered. Notably, if asymmetric divisions correlated with unequal partitioning of actin-related contents in a process linked with embryonic polarity, differences were revealed between AB daughter cells upon their separation. Taken together, these actin-related asymmetric distributions are adding a layer to the complexity of cell fate acquisition mechanisms in the early embryo.

Dauer larvae are a dormant developmental stage in nematodes that is induced by a range of environmental cues. The molecular mechanisms that transduce these cues to regulate dauer entry have been well characterized in Caenorhabditis elegans, whereas those in other nematode species remain unclear. The closest known sibling species of C. elegans, Caenorhabditis inopinata, occupies a distinct ecological niche and shows an extremely low frequency of dauer formation by starvation in laboratory conditions, suggesting that it could serve as a useful comparative model for analyzing dauer-inducing mechanisms. To support such analysis, we generated a fluorescent dauer reporter, Cin-col-183p::mCherry, in C. inopinata based on a previously reported dauer-specific reporter in C. elegans. This reporter showed fluorescence specifically in the pre-dauer and dauer stages, but not in other developmental stages, indicating that it functions as a dauer-specific marker in C. inopinata. Using these marker strains, we compared the responses to high temperature and RNAi-mediated knockdown of insulin/IGF-1 pathway genes (daf-2, age-1, and pdk-1), and found that dauer induction differs mechanistically between C. elegans and C. inopinata. This dauer-specific fluorescent strain will be a useful tool for investigating the diversity of dauer-inducing mechanisms across nematode species. Article SummaryDauer is a dormant developmental stage in nematodes induced by environmental stress. Although its regulation is well studied in Caenorhabditis elegans, the mechanisms in other species remain unclear. Here, we developed a fluorescent dauer reporter, Cin-col-183p::mCherry, in Caenorhabditis inopinata, a close relative of C. elegans. The reporter was specifically expressed in pre-dauer and dauer stages, confirming its usefulness as a dauer marker. Using this strain, we found that responses to high temperature and insulin/IGF-1 pathway gene knockdown differ between C. elegans and C. inopinata. This reporter will help reveal diversity in dauer-inducing mechanisms across nematode species.

The cockroach Blattella germanica possesses panoistic ovaries, in which oocytes lack nurse cells and therefore need to rely on their own transcriptional activity to support embryogenesis. Ovarian development in this species involves the development of a single basal ovarian follicle (BOF) per gonadotropic cycle, a process strictly regulated by endocrine signals, primarily juvenile hormone and ecdysone, which act at both the transcriptional and translational levels. In addition, transcriptional activity in these ovaries is necessary for both regulating and genome protection, and at this level, PIWI-interacting RNAs (piRNAs) play an essential role. Although insect ovaries are known to be particularly rich in piRNAs, their function in ovary maturation is still not well defined. For this purpose, we characterize the piRNA expression dynamics across seven key developmental and reproductive stages, ranging from late nymphal instars to post-vitellogenic adults. piRNA expression in B. germanica shows coordinated fluctuations. Expression remains stable in previtellogenic ovaries, whereas vitellogenic ovaries show pronounced changes. Moreover, vitellogenic ovaries exhibit reduced piRNA diversity due to strong enrichment of a subset of highly expressed piRNAs. Our data show that although piRNAs predominantly map to transposable elements, particularly LINEs, there is a notable increase in gene-derived piRNAs toward the end of the cycle. Our results suggest regulatory roles of piRNAs in modulating both TEs and mRNAs during BOF maturation, likely related to changes in the follicular cell program.

We provide a detailed description of embryonic development in the convict cichlid (Amatitlania nigrofasciata) from fertilization to hatching at 26 {degrees}C, together with a practical staging table anchored to established teleost reference frameworks. Fertilized eggs were obtained by both natural spawning and artificial fertilization. Unfertilized eggs were ovoid and adhesive, surrounded by a chorion and a sticky mucous layer. Early development proceeded, in broad outline, through the teleost sequence of meroblastic discoidal cleavage, blastula, gastrula, segmentation, and organogenesis. The first cleavage occurred at 1.75 hours post-fertilization (hpf), with subsequent cleavages at 30 min intervals, reaching the 64-cell stage at 4.25 hpf. Cleavage up to the 64-cell stage progressed on a timescale broadly comparable to that reported for other cichlids, whereas the interval from the 64-cell stage to early epiboly was relatively short in this species. The high, sphere, and dome stages occurred at 8, 9, and 10 hpf, respectively, with epiboly initiating at the dome stage. At the dome stage, a marginal thickening interpreted as the presumptive embryonic shield became apparent. During early epiboly, the blastoderm showed pronounced spatial heterogeneity: it was consistently thicker and advanced more rapidly on the prospective embryonic axis side, yielding a readily detectable asymmetry. A morphologically distinct embryonic axis became visible at 40-50% epiboly, and epiboly was completed at 28.5 hpf. Notably, somitogenesis began before epiboly completion (first somites at 85-90% epiboly), indicating temporal overlap between late gastrulation and early segmentation. Major organ primordia became apparent during the overlapping segmentation/organogenesis interval, and hatching occurred around 70 hpf. Newly hatched larvae possessed three pairs of adhesive glands. This staging reference enables reproducible developmental sampling and should facilitate future comparative, mechanistic, and experimental work using the convict cichlid.

Rhodnius prolixus is an insect vector of the protozoan Trypanosoma cruzi, the causative agent of debilitating Chagas disease, which is transmitted to humans during blood feeding. Identifying germline markers is a critical step in advancing vector control and transgenic technologies of these medically important insects. Transmission of genetic traits to the next generation requires proper differentiation of the germline that gives rise to gametes. Germline precursors are established during early stages of development as the primordial germ cell (PGC) population. Among the genes required for this process, vasa homologues exert a conserved role in germline specification. Here, we characterize and validate the genomic structure of the R. prolixus Rp-vasa locus and assess its expression during early embryogenesis. We observe widespread Rp-vasa expression in preblastoderm embryos. Later, during the cellular blastoderm and at the beginning of gastrulation, Rp-vasa and Rp-piwi2 expression is restricted to PGCs, morphologically identifiable as a cluster of cells at the posterior of the embryo. We also report, for the first time, the use of R. prolixus regulatory sequences to drive the expression of exogenous genes. We identify the Rp-vasa regulatory region and show that these cis-regulatory sequences are sufficient to drive Cas9 and dsRed expression in the early embryo. Together, these findings demonstrate that Rp-vasa has great potential for use as a PGC marker and as a driver for gene expression in transgenic and gene editing approaches for Triatomine vectors.

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.

Sex-responsive trait development generates much of the phenotypic variation found in natural populations and diversifies rapidly among closely-related taxa. Furthermore, rather than exhibiting equal sexual dimorphism across all traits, organisms are mosaics of tissues that vary in their degree of dimorphism. Yet, how these mosaic patterns are generated remains largely an open question, as sexually dimorphic traits have typically been studied individually in select model systems. In this study, we compare gene regulatory landscapes across five traits that differ in the degree of morphological sexual dimorphism in the bull-headed dung beetle Onthophagus taurus by assaying tissue-specific gene expression and chromatin accessibility at the onset of pupal development when future adult form is specified. We identify a modest number of pleiotropic regulators associated with sex differences across traits, yet uncover a high degree of sex- and trait-specificity in chromatin architecture within developing tissues. We then confirm the role of the sex determination factor doublesex in the regulation of sex differences through expression of sex-specific isoforms, and uncover trait- and sex-specific sets of Doublesex binding sites likely underpinning context specific sexual dimorphisms. Further, we identify and functionally validate the transcription factor ventral veinless as a regulator of sexually dimorphic development. Our findings suggest that in contrast to doublesex, ventral veinless does not exhibit sex-biased expression, yet exerts its sex-specific regulation via sets of differentially accessible binding sites. This work furthers our understanding of the molecular mechanisms instructing the development of sex differences and provides novel insights illustrating how transcriptional activity and chromatin remodeling interact to generate sexual dimorphism in a trait-specific manner. More generally, our work contributes to a growing body of knowledge on how development integrates cues such as sex determination to enable highly similar genomes to yield diverse phenotypic outcomes.

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

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.

Sea star wasting disease has caused widespread mortality in the kelp forest predator, the sunflower sea star (Pycnopodia helianthoides). Wild populations have declined by up to 99% in parts of their native range along the western North American coast. In response, a multi-institutional conservation breeding and rearing program has been initiated to support future reintroduction efforts for the species. We split a full-sibling cohort across four larval density treatments (1 larva/ml, 2 larvae/ml, 5 larvae/ml, and 15-20 larvae/ml) to assess the effects on larval settlement, juvenile survival, and juvenile fitness at 12 months old. Stars raised in the highest density treatment displayed a lower settlement rate and were significantly smaller than the other density groups at 12 months old, but showed no significant difference in flip time, a measure of fitness. Additionally, measurements of diameter, weight, and arm count across modern and historical juvenile and adult stars indicate that P. helianthoides experience exponential weight gain as they grow in length, with corresponding asymptotic growth in arm count. These findings will inform best practices for the aquarium propagation of P. helianthoides and will contribute to broader efforts aimed at reestablishing populations in the wild.