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Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

The evolution of skull diversity in Crocodylia is rather well documented, but the developmental foundation of their cranial architecture remains poorly understood. Here, we present the first three-dimensional reconstruction of the embryonic cartilaginous skull of Caiman crocodilus based on histological sections. We provide a comprehensive anatomical description and morphometric analysis of the chondrocranium of this species, integrating linear measurements and comparative anatomical data to assess interspecific variation and evaluate whether closely related taxa exhibit greater similarity in chondrocranial morphology. We identified both qualitative and quantitative differences between major crocodilian clades, which may reflect diverse ecological demands. Within Crocodylidae, orbitotemporal proportions strongly influence chondrocranial morphology, likely reflecting adaptations related to bite force and visual acuity, linked to prey type and diverse aquatic habits. Within Alligatoridae, by contrast, the emphasis on nasal capsule proportions suggests a reliance on olfactory and respiratory functions, consistent with their occupation of densely vegetated environments and more restricted geographic ranges. Additionally, we identified a set of embryonic diagnostic features located in the splanchnocranium of Caiman and in the neurocranium of Alligatoridae. These findings shed new light on the developmental basis of cranial diversity in Crocodylia and emphasize the evolutionary significance of chondrocranial traits in shaping macroevolutionary patterns.

Abstract Dicroglossid tadpoles are an important element of pond and stream communities in Southeast Asia. They are often found in syntopy but belong to different trophic guilds, from generalized detritophages to specialized predators. In the present research, the external and internal larval morphologies of seven anuran species of the family Dicroglossidae distributed in Vietnam ( Fejervarya limnocharis , Fejervarya moodiei , Hoplobatrachus rugulosus , Limnonectes dabanus , Occidozyga lima , Occidozyga martensii , and Quasipaa verrucospinosa ) are described and compared. An integrated approach is followed in the study by describing external and mouthparts morphologies, gross structure of the digestive tract, and the structure of chondrocranium and hyobranchium, which revealed the main trophic adaptations of tadpoles. Different trends in the evolution of larval macrophagy in Dicroglossidae led to the formation of fundamentally different larval forms. In the first case, the tadpoles are opportunistic predators that retain the ability to use a wide range of food resources ( Hoplobatrachus ); they have extremely specialized mouthparts but moderately transformed digestive tract and larval skeleton. In the other case, tadpoles are highly specialized obligate carnivores ( Occidozyga ) that have completely lost omnivory, with a radically transformed digestive tract, chondrocranium and hyobranchium. A comparison with generalized tadpoles revealed that in dicroglossids, the transformations of the larval skeleton due to carnivory mainly affect the labial cartilages and the palatoquadrate cartilage, as well as the hyobranchium. These transformations are common to macrophagous tadpoles of different taxonomic groups and result in greater larval morphological diversity.

The first record of well-preserved chondrocranial elements in mosasaurids is here described. These elements are preserved in situ in a Coniacian skull found in north-central Colombia, inside a calcareous concretion. Based on a 3D model generated from computed tomography scans, we identified elements of the nasal and orbitotemporal regions. Our descriptions show that in this specimen, the chondrocranium was reduced, more so than in most lacertilians (including their closest recent relatives, the varanids), but not as severely as in snakes or amphisbaenians (which have an extremely reduced chondrocranium and limbs). The new evidence suggests that the reduction in the chondrocranium in mosasaurids could be related to modification of their limbs when adapting to aquatic environments, but also that in mosasaurids, the olfactory tract was reduced, and the optic muscle insertions occurred mainly in the interorbital septum. The exceptional preservation of the chondrocranial elements in the specimen is facilitated by a gray mineralization covering them. XRD analysis and thin section observations indicated that this mineralization is composed of microcrystalline quartz and calcite. We infer that this material was produced by a partial silicification process promoted by lower pH microenvironments associated with bacterial breakdown of non-biomineralized tissues during early diagenesis.

Background The craniofacial skeleton is an evolutionary innovation of vertebrates. Due to its complexity and importance to protect the brain and aid in essential functions (e.g., feeding), its development requires a precisely tuned sequence of chondrification and/or ossification events. The comparison of sequential patterns of cartilage formation bears important insights into the evolution of development. Discoglossus scovazzi is a basal anuran species. The comparison of its chondrocranium (cartilaginous neuro- & viscerocranium) development with other basal anurans ( Xenopus laevis , Bombina orientalis ) will help establishing the ancestral pattern of chondrification sequences in anurans and will serve as basis for further studies to reconstruct ancestral conditions in amphibians, tetrapods, and vertebrates. Furthermore, evolutionary patterns in anurans can be studied in the light of adaptations once the ancestral sequence is established. Results We present a comprehensive overview on the chondrocranium development of D. scovazzi. With clearing and staining, histology and 3D reconstructions we tracked the chondrification of 44 elements from the first mesenchymal Anlagen to the premetamorphic cartilaginous head skeleton and illustrate the sequential changes of the skull. We identified several anuran and discoglossoid traits of cartilage development. In D. scovazzi the mandibular, hyoid, and first branchial arch Anlagen develop first followed by stepwise addition of the branchial arches II, III, and IV. Nonetheless, there is no strict anterior to posterior chondrification pattern within the viscerocranium of D. scovazzi . Single hyoid arch elements chondrify after elements of the branchial arch and mandibular arch elements chondrify after elements of the branchial arch I. Conclusions In Osteichthyes, neurocranial elements develop in anterior to posterior direction. In the anurans investigated so far, as well as in D. scovazzi , the posterior parts of the neurocranium extend anteriorly, while the anterior parts of the neurocranium, extend posteriorly until both parts meet and fuse. Anuran cartilaginous development differs in at least two crucial traits from other gnathostomes which further supports the urgent need for more developmental investigations among this clade to understand the evolution of cartilage development in vertebrates.

In this study, we morphologically examine larval specimens of Ranitomeya variabilis , which were breed in captivity and genetically determined to belong to the French Guiana population. We provide detailed data on the external morphology, chondrocranium, cranial muscle systems and inner organs of the tadpoles. Additionally, we provide essential characteristics for the recognition of the tadpoles of the different Ranitomeya species. The external morphology of the R. variabilis tadpoles was assessed by measurements and photographs of the specimens. Internal morphology was analyzed using µCT images, µCT-based three-dimensional reconstructions, and dissection of specimens for organ and muscle descriptions. The majority of the muscle configurations observed herein for larval specimens of R. variabilis are consistent with data presented in previous studies for larvae of other Ranitomeya species. In addition, several of the observed morphological characters are defined for different taxonomic levels within Dendrobatidae, e.g., the reduction of tectal cartilages and the insertion of the M. rectus cervicis on the third or fourth branchial arch. The absence of the anterolateral process of the ceratohyal in Ranitomeya and Dendrobates further strengthens their close relationship, in contrast to the more distantly related Epipedobates and Phyllobates where this structure is present. The absence of the M. levator arcuum branchialium I and II, the M. interhyoideus posterior and the M. diaphragmatopraecordialis might be defining traits for Ranitomeya . The specific characters observed in this study for R. variabilis , are the fusion of the superficialis and profundus portion of the musculus levator longus larvae and the absence of the parotic crista.

The Rhinella spinulosa group is a clade of toads that inhabit the Andes mountains from northern Ecuador to Patagonia. Its taxonomy was recently revised, and in its new arrangement comprises nine species, including Rhinella gallardoi, traditionally placed in a different intrageneric group. In this work we studied the larval external and internal morphology in this group, by describing for the first time tadpoles of R. achalensis, R. gallardoi, and R. vellardi, and then summarizing morphological data for R. altiperuviana, R. limensis, R. papillosa, R. spinulosa, and R. trifolium. Although we found no diagnostic larval features for the whole clade, two distinct morphs were identified. Most tadpoles were highly pigmented and slender, and their oral discs showed a long gap in the second labial tooth row; conversely, tadpoles of R. limensis and R. vellardi shared a globose body and a very short gap. Buccal and musculoskeletal features were highly conserved within the group and regarding other Rhinella, and included four lingual papillae, nonkeratinized spurs, tripartite suprarostral cartilages, quadrato-orbital commissure, and in musculature, m. subarcualis rectus II–IV invading the branchial septum III and laryngeal muscles reduced or absent.

Elasmobranchs are common, iconic species in public aquaria; their wild counterparts are key members of marine ecosystems. Post-mortem examination is a critical tool for disease monitoring of wild elasmobranchs and for management of those under human care. Careful necropsy of the head, with a focus on clinically relevant anatomy, can ensure that proper samples are collected, increasing the chance of presumptive diagnoses prior to slower diagnostic workup. Immediate feedback from a thorough head necropsy allows for faster management decisions, often identifying pathogens, routes of pathogen entry, and pathogenesis, which are current shortcomings in published literature. This article proposes a protocol for necropsy of the elasmobranch chondrocranium, emphasizing unique anatomy and careful dissection, evaluation, and sampling of the endolymphatic pores and ducts, inner ears, brain, and olfactory system as part of a complete, whole-body necropsy. Extensive use of cytology and microbiology, along with thorough sample collection for histology and molecular biology, has proven effective in identifying a wide range of pathogens and assisting with characterization of pathogenesis. The cause of mortality is often identified from a head necropsy alone, but does not replace a thorough whole-body dissection. This protocol for necropsy and ancillary diagnostic sample collection and evaluation was developed and implemented in the necropsy of 189 wild and aquarium-housed elasmobranchs across 18 species over 13 years (2011–2023) in California. Using this chondrocranial approach, meningoencephalitis was determined to be the primary cause of mortality in 70% (118/168) of stranded wild and aquarium-housed elasmobranchs. Etiology was largely bacterial or protozoal. Carnobacterium maltaromaticum bacterial meningoencephalitis occurred in salmon sharks ( Lamna ditropis ), shortfin mako sharks ( Isurus oxyrinchus ), common thresher sharks ( Alopias vulpinus ), and one Pacific electric ray ( Tetronarce californica ). Miamiensis avidus was the most common cause of protozoal meningoencephalitis and found almost exclusively in leopard sharks ( Triakis semifasciata ) and bat rays ( Myliobatis californica ) that stranded in San Francisco Bay. Bacterial pathogens were found to use an endolymphatic route of entry, while protozoa entered via the nares and olfactory lamellae. Trauma was the second most common cause of mortality and responsible for 14% (24/168) of wild shark strandings and deaths of aquarium-housed animals.

The head represents the most complex part of the body and a distinctive feature of the vertebrate body plan. This intricate structure is assembled during embryonic development in the four-dimensional process of morphogenesis. The head integrates components of the central and peripheral nervous system, sensory organs, muscles, joints, glands, and other specialized tissues in the framework of a complexly shaped skull. The anterior part of the head is referred to as the face, and a broad spectrum of facial shapes across vertebrate species enables different feeding strategies, communication styles, and diverse specialized functions. The face formation starts early during embryonic development and is an enormously complex, multi-step process regulated on a genomic, molecular, and cellular level. In this review, we will discuss recent discoveries that revealed new aspects of facial morphogenesis from the time of the neural crest cell emergence till the formation of the chondrocranium, the primary design of the individual facial shape. We will focus on molecular mechanisms of cell fate specification, the role of individual and collective cell migration, the importance of dynamic and continuous cellular interactions, responses of cells and tissues to generated physical forces, and their morphogenetic outcomes. In the end, we will examine the spatiotemporal activity of signaling centers tightly regulating the release of signals inducing the formation of craniofacial skeletal elements. The existence of these centers and their regulation by enhancers represent one of the core morphogenetic mechanisms and might lay the foundations for intra- and inter-species facial variability.

We compare reproductive features, development, and larval morphology in three closely related species of sticky frogs ( Kalophrynus Tschudi, 1838) inhabiting the lowland and mountain forests of Vietnam and displaying a variety of reproductive modes. While K. interlineatus breeds in open temporary ponds, K. honbaensis and K. cryptophonus are phytotelm-breeders using tree hollows and bamboo stems for reproduction. Their tadpoles also differ in trophic specialization: larval K. interlineatus are typical suspension-feeders, whereas K. honbaensis and K. cryptophonus are obligatorily oophagous. All three species differ in egg and clutch sizes, duration of embryonal period and hatching stage, and the structure of the larval digestive tract and skeleton. Based on external and internal morphology, we conclude that tadpoles of K. interlineatus and K. cryptophonus represent two “extremes” of the adaptive spectrum of microhylid larvae, while K. honbaensis displays a set of transitory traits. Relying on these new findings in anuran biology, we discuss reproductive, ontogenetic, and morphological rearrangements during the transition from pond breeding to phytotelm breeding and from microphagy to macrophagy as well as the significance of the revealed adaptations to different habitats and larval life modes.