Ultrastructure reveals ancestral vertebrate pharyngeal skeleton in yunnanozoans.

Pharyngeal arches are a key innovation that likely contributed to the evolution of the jaws and braincase of vertebrates. It has long been hypothesized that the pharyngeal (branchial) arch evolved from an unjointed cartilaginous rod in vertebrate ancestors such as that in the nonvertebrate chordate amphioxus, but whether such ancestral anatomy existed remains unknown. The pharyngeal skeleton of controversial Cambrian animals called yunnanozoans may contain the oldest fossil evidence constraining the early evolution of the arches, yet its correlation with that of vertebrates is still disputed. By examining additional specimens in previously unexplored techniques (for example, x-ray microtomography, scanning and transmission electron microscopy, and energy dispersive spectrometry element mapping), we found evidence that yunnanozoan branchial arches consist of cellular cartilage with an extracellular matrix dominated by microfibrils, a feature hitherto considered specific to vertebrates. Our phylogenetic analysis provides further support that yunnanozoans are stem vertebrates.

J. P. Hill and Katherine Watson's studies of the neural crest in marsupials.

In the early part of the 20th century, J. P. Hill and K. P. Watson embarked on a comprehensive study of the development of the brain in Australian marsupials. Their work included series from three major groups: dasyurids, peramelids, and diprotodonts, covering early primitive streak through brain closure and folding stages. While the major part of the work was on the development of the brain, in the course of this work they documented that cellular proliferations from the neural plate provided much of the mesenchyme of the branchial arches. These proliferations are now known to be the neural crest. However, except for a very brief note, published shortly after Hill's death, this work was never published. In this study, I present Hill and Watson's work on the development of the early neural plate and the neural crest in marsupials. I compare their findings with published work on the South American marsupial, Monodelphis domestica and demonstrate that patterns reported in Monodelphis are general for marsupials. Further, using their data I demonstrate that in dasyurids, which are ultra-altricial at birth, the neural crest migrates early and in massive quantities, even relative to other marsupials. Finally, I discuss the historical context and speculate on reasons for why this work was unpublished. I find little support for ideas that Hill blocked publication because of loyalty to the germ layer theory. Instead, it appears primarily to have been a very large project that was simply orphaned as Watson and Hill pursued other activities.

Paraneuronal pseudobranchial neurosecretory system in tank goby Glossogobius giuris with special reference to novel neurohaemal contact complex.

Various putative oxygen chemosensory cells are reported to be present throughout the vertebrate body performing pivotal roles in respiration by initiating responses during acute hypoxia. Since air-breathing fishes often are exposed to the oxygen-deficient milieu, in such conditions various chemosensory cells operate in an orchestrated fashion. The Pseudobranchial neurosecretory system (PSNS) a newly discovered system, is one of these. It has been placed in the category of "Diffuse NE systems (DNES)". It is found in all the catfish species and in some other non-catfish group of teleosts. In catfishes, it is present in close association with the carotid labyrinth- a chemosensory structure, known in fish and amphibians. The presence of this system in Glossogobius giuris, in association with the pseudobranch, a structure considered to be precursor of carotid labyrinth, is a significant finding. In an attempt to study the structure and organization of the pseudobranchial neurosecretory system in a non-catfish species of teleost, the present investigation was undertaken on a goby G. giuris. The histological observations, using a neurosecretion-specific stain, revealed the presence of this system in G. giuris. The findings are discussed in the light of the association of PSNS with pseudobranch and the type of "neurohaemal contact complex" formed between this neurosecretory system and the elements of the circulatory system.

Sequence of chondrocranial development in the oriental fire bellied toad Bombina orientalis.

The vertebrate head as a major novelty is directly linked to the evolutionary success of the vertebrates. Sequential information on the embryonic pattern of cartilaginous head development are scarce, but important for the understanding of its evolution. In this study, we use the oriental fire bellied toad, Bombina orientalis, a basal anuran to investigate the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen in post-neurulation stages until the premetamorphic larvae. We use different methodological approaches like classic histology, clearing and staining, and antibody staining to examine the larval skeletal morphology. Our results show that in contrast to other vertebrates, the ceratohyals are the first centers of chondrification. They are followed by the palatoquadrate and the basihyal. The latter later fuses to the ceratohyal and the branchial basket. Anterior elements like Meckel's cartilage and the rostralia are delayed in development and alter the ancestral anterior posterior pattern observed in other vertebrates. The ceratobranchials I-IV, components of the branchial basket, follow this strict anterior-posterior pattern of chondrification as reported in other amphibians. Chondrification of different skeletal elements follows a distinct pattern and the larval skeleton is nearly fully developed at Gosner Stage 28. We provide baseline data on the pattern and timing of early cartilage development in a basal anuran species, which may serve as guidance for further experimental studies in this species as well as an important basis for the understanding of the evolutionary changes in head development among amphibians and vertebrates.

A reappraisal and revision of the numbering of the pharyngeal arches.

The pharyngeal arches are a prominent and significant feature of vertebrate embryos. These are visible as a series of bulges on the lateral surface of the embryonic head. In humans, and other amniotes, there are five pharyngeal arches numbered 1, 2, 3, 4 and 6; note the missing '5'. This is the standard scheme for the numbering of these structures, and it is a feature of modern anatomy textbooks. In this article, we discuss the rationale behind this odd numbering, and consider its origins. One reason given is that there is a transient 5th arch that is never fully realized, while another is that this numbering reflects considerations from comparative anatomy. We show here, however, that neither of these reasons has substance. There is no evidence from embryology for a '5th' arch, and the comparative argument does not hold as it does not apply across the vertebrates. We conclude that there is no justification for this strange numbering. We suggest that the pharyngeal arches should simply be numbered 1, 2, 3, 4 and 5 as this would be in keeping with the embryology and with the general numbering of the pharyngeal arches across the vertebrates.

Multiethnic GWAS Reveals Polygenic Architecture of Earlobe Attachment.

The genetic basis of earlobe attachment has been a matter of debate since the early 20th century, such that geneticists argue both for and against polygenic inheritance. Recent genetic studies have identified a few loci associated with the trait, but large-scale analyses are still lacking. Here, we performed a genome-wide association study of lobe attachment in a multiethnic sample of 74,660 individuals from four cohorts (three with the trait scored by an expert rater and one with the trait self-reported). Meta-analysis of the three expert-rater-scored cohorts revealed six associated loci harboring numerous candidate genes, including EDAR, SP5, MRPS22, ADGRG6 (GPR126), KIAA1217, and PAX9. The large self-reported 23andMe cohort recapitulated each of these six loci. Moreover, meta-analysis across all four cohorts revealed a total of 49 significant (p < 5 × 10-8) loci. Annotation and enrichment analyses of these 49 loci showed strong evidence of genes involved in ear development and syndromes with auricular phenotypes. RNA sequencing data from both human fetal ear and mouse second branchial arch tissue confirmed that genes located among associated loci showed evidence of expression. These results provide strong evidence for the polygenic nature of earlobe attachment and offer insights into the biological basis of normal and abnormal ear development.

The development of the cucullaris muscle and the branchial musculature in the Longnose Gar, (Lepisosteus osseus, Lepisosteiformes, Actinopterygii) and its implications for the evolution and development of the head/trunk interface in vertebrates.

The vertebrate head/trunk interface is the region of the body where the different developmental programs of the head and trunk come in contact. Many anatomical structures that develop in this transition zone differ from similar structures in the head or the trunk. This is best exemplified by the cucullaris/trapezius muscle, spanning the head/trunk interface by connecting the head to the pectoral girdle. The source of this muscle has been claimed to be either the unsegmented head mesoderm or the somites of the trunk. However most recent data on the development of the cucullaris muscle are derived from tetrapods and information from actinopterygian taxa is scarce. We used classical histology in combination with fluorescent whole-mount antibody staining and micro-computed tomography to investigate the developmental pattern of the cucullaris and the branchial muscles in a basal actinopterygian, the Longnose gar (Lepisosteus osseus). Our results show (1) that the cucullaris has been misidentified in earlier studies on its development in Lepisosteus. (2) Cucullaris development is delayed compared to other head and trunk muscles. (3) This developmental pattern of the cucullaris is similar to that reported from some tetrapod taxa. (4) That the retractor dorsalis muscle of L. osseus shows a delayed developmental pattern similar to the cucullaris. Our data are in agreement with an explanatory scenario for the cucullaris development in tetrapods, suggesting that these mechanisms are conserved throughout the Osteichthyes. Furthermore the developmental pattern of the retractor dorsalis, also spanning the head/trunk interface, seems to be controlled by similar mechanisms.

A three-dimensional placoderm (stem-group gnathostome) pharyngeal skeleton and its implications for primitive gnathostome pharyngeal architecture.

The pharyngeal skeleton is a key vertebrate anatomical system in debates on the origin of jaws and gnathostome (jawed vertebrate) feeding. Furthermore, it offers considerable potential as a source of phylogenetic data. Well-preserved examples of pharyngeal skeletons from stem-group gnathostomes remain poorly known. Here, we describe an articulated, nearly complete pharyngeal skeleton in an Early Devonian placoderm fish, Paraplesiobatis heinrichsi Broili, from Hunsrück Slate of Germany. Using synchrotron light tomography, we resolve and reconstruct the three-dimensional gill arch architecture of Paraplesiobatis and compare it with other gnathostomes. The preserved pharyngeal skeleton comprises elements of the hyoid arch (probable ceratohyal) and a series of branchial arches. Limited resolution in the tomography scan causes some uncertainty in interpreting the exact number of arches preserved. However, at least four branchial arches are present. The final and penultimate arches are connected as in osteichthyans. A single median basihyal is present as in chondrichthyans. No dorsal (epibranchial or pharyngobranchial) elements are observed. The structure of the pharyngeal skeleton of Paraplesiobatis agrees well with Pseudopetalichthys from the same deposit, allowing an alternative interpretation of the latter taxon. The phylogenetic significance of Paraplesiobatis is considered. A median basihyal is likely an ancestral gnathostome character, probably with some connection to both the hyoid and the first branchial arch pair. Unpaired basibranchial bones may be independently derived in chondrichthyans and osteichthyans.

Morphological novelty and modest developmental truncation in Barboides, Africa's smallest vertebrates (Teleostei: Cyprinidae).

Miniaturization, the evolution of extremely small adult body size, is widespread amongst animals and commonly associated with novel ecological, physiological, and morphological attributes. The phenotypes of miniaturized taxa are noteworthy because they combine reductions and structural simplifications with novel traits not developed in their larger relatives. Previous research on miniature cyprinid fishes (focused predominantly on South and South East Asian taxa of a single subfamily) has identified two distinct classes of miniature taxa: proportioned dwarves and developmentally truncated miniatures. Miniaturization has also occurred independently in the subfamily Cyprininae, particularly in African lineages. We investigate the skeletal anatomy of Barboides, a genus of miniature African cyprinids that includes Africa's smallest known species of vertebrates, to assess whether miniaturization has resulted in similar organismal outcomes in different lineages of the Cyprinidae. The skeleton of Barboides is characterized by the complete absence of a number of dermal and endochondral ossifications, and marked reduction in size and/or complexity of other skeletal elements, particularly those of the dermatocranium. Absent skeletal elements in Barboides include those which develop relatively late in the ossification sequence of the non-miniature African relative 'Barbus' holotaenia suggesting that their absence in Barboides can be explained by a simple scenario of developmental truncation. In contrast to this theme of loss and reduction, the os suspensorium of Barboides is enlarged and the outer arm distally trifid and associated with a novel bulbous muscle in males. An evaluation of the skeleton of Barboides provides further evidence for a link between developmental truncation and evolutionary morphological novelty in Cyprinidae. In the spectrum of miniature cyprinids ranging from proportioned dwarves with few bones missing to highly progenetic taxa with dozens of missing bones, the two species of Barboides range roughly in the middle showing that the extremes are connected by intermediate levels of truncatedness.

Morphology and relationships of ultimobranchial body with pharyngeal pouches

The ultimobranchial body in humans is still controversial and different theories have been put forward. The aim of this study was to clarify the topographical anatomy of the ultimobranchial body and surrounding tissue during early development. Human embryos at 5-7 weeks of development were used for morphological observation. During the early stages, the sections displayed a ladder-like arrangement of the second to fourth endodermal pouches. The fourth pouch was located in front of the nodosa ganglion. The bilateral fifth pharyngeal pouches protruded anterolaterally to form a Ushaped lumen surrounding the arytenoid swelling. During the middle stages, the third pharyngeal pouch was identified near the fourth pharyngeal artery and the fourth pharyngeal pouch was located anterior to the parathyroid gland IV. We identified a cyst-like structure that is composed of a cell cluster facing to a small lumen as the ultimobranchial body. During the late stages, the lateral thyroid arising from the fourth pharyngeal pouch was located medial to the common carotid artery and joined to dorsal surface of the thyroid gland anlage. The thymus anlage arising from the third pharyngeal pouch was an independent structure that was located lateral to the common carotid artery. However, the ultimobranchial body had disappeared and did not integrate in the thyroid gland. We concluded that (1) the thymus originates from the third pharyngeal pouch; (2) the lateral thyroid originates from the fourth pharyngeal pouch; (3) the ultimobranchial body originates from the fifth pharyngeal pouch

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The hyoid arch and braincase anatomy of Acanthodes support chondrichthyan affinity of 'acanthodians'.

Solving the evolutionary relationships of the acanthodians is one of the key problems in reconstructing ancestral anatomical conditions for the jawed vertebrates (gnathostomes). Current debate concerns whether acanthodians are an assemblage of stem chondrichthyans, or a more generalized grade encompassing some early stem osteichthyans. The skull anatomy of Acanthodes bronni has been pivotal in these debates, owing to tension between chondrichthyan- and osteichthyan-like models of reconstruction. We use computed tomography scanning and traditional palaeontological techniques to resolve the long-standing debate about the anatomy of the jaw suspension. We establish the correct length of the hyomandibula and show that it attaches to a process on the ventrolateral angle of the braincase below the jugular vein groove. This condition corresponds precisely to that in chondrichthyans. This character represents an unambiguously optimized synapomorphy with chondrichthyans given current gnathostome phylogenies, corroborating the growing consensus of the chondrichthyan affinity of acanthodians.

A new heart for a new head in vertebrate cardiopharyngeal evolution.

It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts - both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.

Anatomical and physiological studies of bigheaded carps demonstrate that the epibranchial organ functions as a pharyngeal taste organ.

The epibranchial organ (EO) is an enigmatic tubular organ found in the pharyngeal cavity of many filter-feeding fishes. We investigated whether it might function as a taste organ that mediates aggregation and ingestion of planktonic food within the buccal cavity. The EO and associated structures of bighead and silver carps, two successful and invasive planktivorous fishes, were examined using histological and electrophysiological techniques. Both species possess finely structured gill rakers that extend directly via a series of protrusions into each of the four blind canals which are organized as the muscular EO, suggesting that the gill rakers and EO probably function in an integrated manner. Both the interior and exterior surfaces of the EOs of both species are covered with high densities of taste buds and solitary chemosensory cells (SCCs) as well as mucous cells. Conversely, taste buds are scarce in both the buccal cavities and external portions of the head and mouth of both species. Electrophysiological recordings from a caudal branch of the vagus nerve (cranial nerve X) found to innervate the EO showed it to be sensitive to chemicals found in a planktonic diet. l-Amino acids accounted for some, but not all of the neural activity. We conclude that taste buds and SCCs located on the EO and gill rakers probably serve to chemically detect food particles, which the EO then aggregates by mucus secretion before eventually expelling them onto the floor of the pharynx for ingestion. This specialized, pharyngeal chemosensory structure may explain the feeding success of these, and perhaps other planktivorous, filter-feeding fishes.

A Palaeozoic shark with osteichthyan-like branchial arches.

The evolution of serially arranged, jointed endoskeletal supports internal to the gills--the visceral branchial arches--represents one of the key events in early jawed vertebrate (gnathostome) history, because it provided the morphological basis for the subsequent evolution of jaws. However, until now little was known about visceral arches in early gnathostomes, and theories about gill arch evolution were driven by information gleaned mostly from both modern cartilaginous (chondrichthyan) and bony (osteichthyan) fishes. New fossil discoveries can profoundly affect our understanding of evolutionary history, by revealing hitherto unseen combinations of primitive and derived characters. Here we describe a 325 million year (Myr)-old Palaeozoic shark-like fossil that represents, to our knowledge, the earliest identified chondrichthyan in which the complete gill skeleton is three-dimensionally preserved in its natural position. Its visceral arch arrangement is remarkably osteichthyan-like, suggesting that this may represent the common ancestral condition for crown gnathostomes. Our findings thus reinterpret the polarity of some arch features of the crown jawed vertebrates and invert the classic hypothesis, in which modern sharks retain the ancestral condition. This study underscores the importance of early chondrichthyans in resolving the evolutionary history of jawed vertebrates.

Muscular system in the pacific bluefin tuna Thunnus orientalis (Teleostei: Scombridae).

The muscular system in the Pacific bluefin tuna Thunnus orientalis is studied in detail. For the first time, a complete description of the muscular anatomy of a thunnid is provided here. Eighty-two elements including subdivisions of components of the muscular system are identified. This is less than found in a basal perciform and two other investigated scombrid species, owing mainly to the absence or fusion of pectoral, pelvic and caudal fin muscles. The absence of elements of the basal perciform pattern was most prominent in the caudal fin, which includes only the flexor dorsalis, flexor ventralis, hypochordal longitudinalis, and interradialis. In the caudal fin, the medial fan-shaped ray was identified as the first dorsal ray, judging from myological and neuroanatomical characters. The highly developed gill filament muscles in Thunnus orientalis and sheet-like rectus communis control gill ventilation. Long body muscle tendons reduce the metabolic energy needed during rapid and continuous swimming. These characters are interpreted as adaptations in the context of the oceanic life style of the species.

Particle retention in suspension-feeding fish after removal of filtration structures.

The suspension-feeding cichlids Oreochromis aureus (blue tilapia) and Oreochromis esculentus (ngege tilapia) are able to selectively retain small food particles. The gill rakers and microbranchiospines of these species have been assumed to function as filters. However, surgical removal of these oral structures, which also removed associated mucus, did not significantly affect the total number of 11-200 µm particles ingested by the fish. This result supports the hypothesis that the branchial arch surfaces themselves play an important role in crossflow filtration. Both species selectively retained microspheres greater than 50 µm with gill rakers and microbranchiospines intact as well as removed, demonstrating that neither these structures nor mucus are necessary for size selectivity to occur during biological crossflow filtration. After removal of the gill rakers and microbranchiospines, O. esculentus retained significantly more microspheres 51-70 µm in diameter and fewer 91-130 µm microspheres compared to retention with intact structures, but the particle size selectivity of O. aureus was not affected significantly. These results support conclusions from previous computational fluid dynamics simulations indicating that particle size can have marked effects on particle trajectory and retention inside the fish oropharyngeal cavity during crossflow filtration. The substantial inter-individual variability in particle retention by suspension-feeding fish is an unexplored area of research with the potential to increase our understanding of the factors influencing particle retention during biological filtration.

Homology of the fifth epibranchial and accessory elements of the ceratobranchials among gnathostomes: insights from the development of ostariophysans.

Epibranchials are among the main dorsal elements of the gill basket in jawed vertebrates (Gnathostomata). Among extant fishes, chondrichthyans most resemble the putative ancestral condition as all branchial arches possess every serially homologous piece. In osteichthyans, a primitive rod-like epibranchial 5, articulated to ceratobranchial 5, is absent. Instead, epibranchial 5 of many actinopterygians is here identified as an accessory element attached to ceratobranchial 4. Differences in shape and attachment of epibranchial 5 in chondrichthyans and actinopterygians raised suspicions about their homology, prompting us to conduct a detailed study of the morphology and development of the branchial basket of three ostariophysans (Prochilodus argenteus, Characiformes; Lophiosilurus alexandri and Pseudoplatystoma corruscans, Siluriformes). Results were interpreted within a phylogenetic context of major gnathostome lineages. Developmental series strongly suggest that the so-called epibranchial 5 of actinopterygians does not belong to the epal series because it shares the same chondroblastic layer with ceratobranchial 4 and its ontogenetic emergence is considerably late. This neomorphic structure is called accessory element of ceratobranchial 4. Its distribution among gnathostomes indicates it is a teleost synapomorphy, occurring homoplastically in Polypteriformes, whereas the loss of the true epibranchial 5 is an osteichthyan synapomorphy. The origin of the accessory element of ceratobranchial 4 appears to have occurred twice in osteichthyans, but it may have a single origin; in this case, the accessory element of ceratobranchial 4 would represent a remnant of a series of elements distally attached to ceratobranchials 1-4, a condition totally or partially retained in basal actinopterygians. Situations wherein a structure is lost while a similar neomorphic element is present may lead to erroneous homology assessments; these can be avoided by detailed morphological and ontogenetic investigations interpreted in the light of well-supported phylogenetic hypotheses.

Developmental evidence for serial homology of the vertebrate jaw and gill arch skeleton.

Gegenbaur's classical hypothesis of jaw-gill arch serial homology is widely cited, but remains unsupported by either palaeontological evidence (for example, a series of fossils reflecting the stepwise transformation of a gill arch into a jaw) or developmental genetic data (for example, shared molecular mechanisms underlying segment identity in the mandibular, hyoid and gill arch endoskeletons). Here we show that nested expression of Dlx genes--the 'Dlx code' that specifies upper and lower jaw identity in mammals and teleosts--is a primitive feature of the mandibular, hyoid and gill arches of jawed vertebrates. Using fate-mapping techniques, we demonstrate that the principal dorsal and ventral endoskeletal segments of the jaw, hyoid and gill arches of the skate Leucoraja erinacea derive from molecularly equivalent mesenchymal domains of combinatorial Dlx gene expression. Our data suggest that vertebrate jaw, hyoid and gill arch cartilages are serially homologous, and were primitively patterned dorsoventrally by a common Dlx blueprint.

Comparative myology of the mandibular and hyoid arches of sharks of the order hexanchiformes and their bearing on its monophyly and phylogenetic relationships (Chondrichthyes: Elasmobranchii).

The order Hexanchiformes currently comprises two families, Chlamydoselachidae (frilled sharks) and Hexanchidae (six- and seven-gill sharks), but its monophyly and relationships with other elasmobranchs are still discussed. Previous studies of hexanchiforms addressing these issues were based mainly on external morphology, teeth, skeletal features, and molecular data, whereas the employment of characters derived from variations in muscles has not been significantly explored. Dissections of four species of Hexanchiformes (including Chlamydoselachus anguineus) are reported here describing the mandibular (musculus adductor mandibulae dorsalis, m. adductor mandibulae ventralis, m. levator labii superioris, m. intermandibularis, and m. constrictor dorsalis) and hyoidean (m. constrictor hyoideus dorsalis and ventralis) arch muscles. Our results provide new data concerning the relationships of hexanchiforms to other elasmobranchs. The m. adductor mandibulae superficialis is described and illustrated in C. anguineus, contradicting previous accounts in which is was considered absent. The anteroposterior orientation of the m. adductor mandibulae superficialis in Chlamydoselachus is similar to the pattern found in hexanchids, squaloids, and hypnosqualeans (including batoids), suggesting it was secondarily lost in Echinorhinus. This muscle therefore provides further support for the inclusion of the Chlamydoselachidae and Hexanchidae in the Squalomorphi, and not basal to all other elasmobranchs or nested within an all-shark collective, as has been previously proposed. However, the m. adductor mandibulae superficialis originating at the jaw joint and with an aponeurotic insertion in hexanchids, squaliforms, and hypnosqualeans, may be a separate derived feature uniting these taxa. The insertion of the m. constrictor dorsalis is restricted to the postorbital articulation in hexanchids, whereas it extends farther anteriorly in C. anguineus. The insertion of the m. constrictor hyoideus dorsalis solely on the palatoquadrate is found exclusively in the Hexanchidae. We conclude that no specific pattern of mandibular or hyoid arch muscles support the monophyly of hexanchiforms (i.e., including Chlamydoselachus).