Cranial ontogeny in Philautus silus (Anura: Ranidae: Rhacophorinae) reveals few similarities with other direct-developing anurans

Direct development has evolved in rhacophorine frogs independently from other anuran lineages, thereby offering an opportunity to assess features associated with this derived life history. Using a developmental series of the direct-developing Philautus silus (Ranidae: Rhacophorinae) from Sri Lanka, we examine features of cranial morphology that are part of a suite of adaptations that facilitate feeding in free-living tadpoles, but have been changed or lost in other direct-developing lineages. Larval-specific upper jaw cartilages, which are absent from many non-rhacophorine direct-developing species (such as Eleutherodactylus coqui), develop in embryos of P. silus. Similarly, lower jaw cartilages initially assume a larval morphology, which is subsequently remodeled into the adult jaw configuration before hatching. However, the cartilaginous jaw suspension and hyobranchial skeleton never assume a typical larval morphology. The palatoquadrate, which suspends the lower jaw, lacks the posterior connections to the braincase found in many metamorphosing species. Unlike in metamorphosing species, bone formation in P. silus begins before hatching. However, the sequence of bone formation resembles that of metamorphosing anurans more than that of other direct developers. In particular, P. silus does not exhibit precocious ossification of the lower jaw, which is characteristic of some frogs and caecilians that lack a free-living tadpole. These data reveal some similarities between Philautus and other direct-developing anurans. However, the departure of Philautus embryos from the generalized tadpole skeletal morphology is less pronounced than that observed in other direct-developing taxa.     

Jaw muscle development as evidence for embryonic repatterning in direct-developing frogs.

The Puerto Rican direct-developing frog Eleutherodactylus coqui (Leptodactylidae) displays a novel mode of jaw muscle development for anuran amphibians. Unlike metamorphosing species, several larval-specific features never form in E. coqui; embryonic muscle primordia initially assume an abbreviated, mid-metamorphic configuration that is soon remodelled to form the adult morphology before hatching. Also lacking are both the distinct population of larval myofibres and the conspicuous, larval-to-adult myofibre turnover that are characteristic of muscle development in metamorphosing species. These modifications are part of a comprehensive alteration in embryonic cranial patterning that has accompanied life history evolution in this highly speciose lineage. Embryonic ''repatterning'' in Eleutherodactylus may reflect underlying developmental mechanisms that mediate the integrated evolution of complex structures. Such mechanisms may also facilitate, in organisms with a primitively complex life cycle, the evolutionary dissociation of embryonic, larval, and adult features.     

Cranial neural crest-cell migration in the direct-developing frog, Eleutherodactylus coqui: molecular heterogeneity within and among migratory streams

Direct development is a specialized reproductive mode that has evolved repeatedly in many different lineages of amphibians, especially anurans. A fully formed, albeit miniature adult hatches directly from the egg; there is no free-living larva. In many groups, the evolution of direct development has had profound consequences for cranial development and morphology, including many components that are derived from the embryonic neural crest. Yet, the developmental bases of these effects remain poorly known. In order to more fully characterize these changes, we used three molecular markers to analyze cranial neural crest-cell emergence and migration in the direct-developing frog, Eleutherodactylus coqui: HNK-1 immunoreactivity, Dlx protein expression, and cholinesterase activity. Our study validates and extends earlier results showing that the comprehensive changes in embryonic cranial patterning, differentiation, and developmental timing that are associated with direct development in Eleutherodactylus have not affected gross features of cranial neural crest biology: the relative timing of crest emergence and the number, configuration and identity of the principal migratory streams closely resemble those seen in metamorphic anurans. The three markers are variably expressed within and among neural crest-cell populations. This variation suggests that determination of cranial neural crest-cells may already have begun at or soon after the onset of migration, when the cells emerge from the neural tube. It is not known how or even if this variation correlates with differential cell lineage or fate. Finally, although HNK-1 expression is widely used to study neural crest migration in teleost fishes and amniotes, E. coqui is the only amphibian known in which it effectively labels migrating neural crest-cells. There are not enough comparative data to determine whether this feature is functionally associated with direct development or is instead unrelated to reproductive mode.     

Loss of ectodermal competence for lateral line placode formation in the direct developing frog Eleutherodactylus coqui.

In the direct-developing frog Eleutherodactylus coqui neuromasts and ganglia of the lateral line system never develop. We show here that this absence of the lateral line system, which is evolutionarily derived in anurans, is due to very early changes in development. Ectodermal thickenings, which are typical of lateral line placodes, and from which neuromasts and ganglion cells of the lateral line originate, never form in E. coqui, although other neurogenic placodes are present. Moreover, although NeuroD is expressed in the lateral line placodes of Xenopus laevis, corresponding expression sites are lacking in E. coqui. Heterospecific transplantation experiments show that axolotl ectoderm can be induced to form lateral line placodes after transplantation to E. coqui hosts but that E. coqui ectoderm does not form lateral line placodes on axolotl hosts. This suggests that the loss of the lateral line system in E. coqui is due to the specific loss of ectodermal competence to form lateral line placodes in response to inductive signals. Our results (1) indicate that the competence for lateral line placode formation is distinct and dissociable from the competence to form other neurogenic placodes and (2) support the idea that the lateral line system acts as a module in development and evolution.     

Heterochronies in the cranial development of Asian tree frogs (Amphibia: Anura: Rhacophoridae) with different life histories

The development of bony skull was studied in four species of Asian tree frogs (Rhacophoridae) with different life histories: biphasic development with free larval stage and direct development. In biphasic rhacophorids the sequence of the appearance of cranial bones generally followed the generalized pattern of craniogenesis, which was described for most studied anurans. In contrast, direct-developing species displayed some heterochronies in the formation of skull bones, namely, the accelerated formation of the anlagen of jaw and suspensorium bones. The obtained results support that the embryonization in amphibians is regularly accompanied by a heterochronic repatterning of craniogenesis, rather similar in different phyletic groups.     

Cranial muscle development in frogs with different developmental modes: Direct development versus biphasic development

Normal development in anurans includes a free swimming larva that goes through metamorphosis to develop into the adult frog. We have investigated cranial muscle development and adult cranial muscle morphology in three different anuran species. Xenopus laevis is obligate aquatic throughout lifetime, Rana (Lithobates) pipiens has an aquatic larvae and a terrestrial adult form, and Eleutherodactylus coqui has direct developing juveniles that hatch from eggs deposited on leaves (terrestrial). The adult morphology shows hardly any differences between the investigated species. Cranial muscle development of E. coqui shows many similarities and only few differences to the development of Rana (Lithobates) and Xenopus. The differences are missing muscles of the branchial arches (which disappear during metamorphosis of biphasic anurans) and a few heterochronic changes. The development of the mandibular arch (adductor mandibulae) and hyoid arch (depressor mandibulae) muscles is similar to that observed in Xenopus and Rana (Lithobates), although the first appearance of these muscles displays a midmetamorphic pattern in E. coqui. We show that the mix of characters observed in E. coqui indicates that the larval stage is not completely lost even without a free swimming larval stage. Cryptic metamorphosis is the process in which morphological changes in the larva/embryo take place that are not as obvious as in normal metamorphosing anurans with a clear biphasic lifestyle. During cryptic metamorphosis, a normal adult frog develops, indicating that the majority of developmental mechanisms towards the functional adult cranial muscles are preserved. J. Morphol. 275:398–413, 2014. © 2013 Wiley Periodicals, Inc.     

Development of the nasal chemosensory organs in two terrestrial anurans: the directly developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), and the metamorphosing toad, Bufo americanus (Anura: Bufonidae)

Nearly all vertebrates possess an olfactory organ but the vomeronasal organ is a synapomorphy for tetrapods. Nevertheless, it has been lost in several groups of tetrapods, including aquatic and marine animals. The present study examines the development of the olfactory and vomeronasal organs in two terrestrial anurans that exhibit different developmental modes. This study compares the development of the olfactory and vomeronasal organs in metamorphic anurans that exhibit an aquatic larva (Bufo americanus) and directly developing anurans that have eliminated the tadpole (Eleutherodactylus coqui). The olfactory epithelium in larval B. americanus is divided into dorsal and ventral branches in the rostral and mid-nasal regions. The larval olfactory pattern in E. coqui has been eliminated. Ontogeny of the olfactory system in E. coqui embryos starts to vary substantially from the larval pattern around the time of operculum development, the temporal period when the larval stage is hypothesized to have been eliminated. The nasal anatomy of the two frogs does not appear morphologically similar until the late stages of embryogenesis in E. coqui and the terminal portion of metamorphosis in B. americanus. Both species and their respective developing offspring, aquatic tadpoles and terrestrial egg/embryos, possess a vomeronasal organ. The vomeronasal organ develops at mid-embryogenesis in E. coqui and during the middle of the larval period in B. americanus, which is relatively late for neobatrachians. Development of the vomeronasal organ in both frogs is linked to the developmental pattern of the olfactory system. This study supports the hypothesis that the most recent common ancestor of tetrapods possessed a vomeronasal organ and was aquatic, and that the vomeronasal organ was retained in the Amphibia, but lost in some other groups of tetrapods, including aquatic and marine animals.     

Metamorphosis and evolution of feeding behaviour in salamanders of the family Plethodontidae

Plethodontid salamanders capture prey with enhanced tongue protraction relative to other salamander taxa, yet metamorphosing plethodontids are hypothesized to be constrained relative to direct-developing plethodontids in their degree of tongue evolution (protraction length and velocity) by the presence of a larval stage in development. In this biphasic life history the hyobranchial apparatus serves the conflicting functions of larval suction feeding and adult tongue protraction. The deletion of the larval stage removes one of the conflicting functions and has thus permitted direct-developing plethodontids to circumvent this constraint and evolve extremely long tongues, which in some species can be projected to 80% of body length. To evaluate this constraint hypothesis and explore taxonomic diversity of feeding behaviours, we studied feeding in larvae, adults and metamorphosing individuals of seven species of metamorphosing plethodontids from the basal taxa Desmognathinae and Hemidactyliini using direct observations, high-speed videography and kinematic analysis. We found that larval plethodontids suction feed, but feeding is suspended entirely during metamorphosis, and aquatic adults do not suction feed. Adults have exapted the terrestrial modes of tongue and jaw prehension for aquatic prey capture. These findings substantiate the premise that suction feeding and tongue protraction are conflicting functions, and thus our results support the constraint hypothesis. Plethodontid adults have evolved their extreme tongue protraction ability at the expense of adult suction feeding. The rapid metamorphosis that characterizes plethodontids may be an adaptation that minimizes the non-feeding period imposed by the evolution of derived tongue protraction in adults. © 2002 The Linnean Society of London, Zoological Journal of the Linnean Society , 2002, 134 , 375–400.     

The morphology and post-hatching development of the skull of Bolitoglossa nicefori (Caudata: Plethodontidae): developmental implications of recapitulation and repatterning

The cranial morphology of the direct-developing salamander Bolitoglossa nicefori and its post-hatching development are described and compared with that of other urodeles. Four stages of cranial development are defined on the basis of conspicuous events that occur during post-hatching ontogeny. The adult skull morphology of B. nicefori is similar to that of other plethodontids; however, some regions show interspecific variation. The post-hatching ontogeny of the skull and the stage of ossification observed in the hatchlings of B. nicefori show two important ontogenetic features: (1) a mosaic of early larval, metamorphic and post-metamorphic skull features in hatchlings, and (2) absence of characteristic larval elements in skull and hyoid apparatus. The distinctive stage of ossification in the hatchlings of B. nicefori could be caused by heterochronic changes in the ossification sequence, compared to the ontogeny of metamorphic salamanders. The possible heterochronic changes and the absence of larval traits are perhaps due to ontogenetic repatterning, yet without an obvious impact on the adult skull morphology (absence of morphological novelties). This might indicate a compartmentalized development. Further studies should be performed in order to establish the possible occurrence of recapitulatory patterns or ontogenetic repatterning in the skull morphogenesis of B. nicefori during its embryonic development.     

Opercular development and ontogenetic re-organization in a direct-developing frog

The direct-developing frog, Eleutherodactylus coqui, has eliminated the tadpole stage from its ontogeny, and lacks many larval characters. We demonstrate that the dermal folds of E. coqui are homologous with the opercular folds of metamorphosing frogs. In both E. coqui and its metamorphic counterparts the opercular folds grow over the developing forelimb before perforating to free the entrapped limb. Opercular perforation in E. coqui occurs even in the absence of the forelimb but shows no signs of thyroid hormone dependence. The condensation of E. coqui development appears due to the excision of the extended larval period of developmental stasis. Analysis of opercular development, when viewed in conjunction with other developmental characters, suggests the ontogenetic period in the ancestral Eleutherodactylus life-history from which the tadpole was likely eliminated. Received: 7 October 1999 / Accepted: 17 January 2000     

Frogs without polliwogs: evolution of anuran direct development

Direct development is the assumption of the adult morphology without progression through an intervening, morphologically distinct, free-living larval phase. We discuss the ecological factors contributing to the evolution of this derived life-history strategy in frogs, and the developmental modifications that facilitate such an unusual mode of embryogenesis. Studies on the Puerto Rican tree frog, Eleutherodactylus coqui, have identified several such modifications, including developmental adaptations for dealing with increased egg size, and loss of tadpole structures. Surprisingly, this direct developer still undergoes a thyroid hormone-dependent metamorphosis, which occurs before hatching. We suggest how the ancestral biphasic developmental pattern may have been rearranged during the evolution of direct development.     

Segmentation of the vertebrate skull: neural-crest derivation of adult cartilages in the clawed frog, Xenopus laevis

We utilize a novel, transgenic cell-labeling system to assess the embryonic derivation of cartilages in the post-metamorphic skull of anuran amphibians. Many of these cartilages form de novo at metamorphosis and have no obvious precursors within the larval skeleton. Most adult cartilages are derived from mandibular- or hyoid-stream neural crest, either individually or in combination; branchial-stream neural crest makes a modest contribution. Each stream also contributes to at least one cartilage in the middle ear or external ear. Four cartilages are composite elements; each is derived from at least two distinct cell populations. Many boundaries between adjacent neural-crest territories are cryptic insofar as they do not coincide with anatomical boundaries. The system of adult cranial segmentation revealed by these fate-mapping results differs in important respects from both the segmentation of the ontogenetically earlier larval skull and the cranial segmentation in amniotes. Most striking is the rostral "inversion" of neural-crest-derived cartilages in Xenopus, such that mandibular stream-derived elements are deployed caudal to those derived from the hyoid stream, which predominate anteriorly. This novel pattern of rostral segmentation may be a consequence of the complex, biphasic life history that is characteristic of most species of living amphibians, and especially anurans, in which cranial architecture is significantly reconfigured at metamorphosis. Neural-crest derivation of the vertebrate skull is not invariant; instead, embryonic derivation of individual components of the cranial skeleton may vary widely among species.     

Limb development in a "nonmodel" vertebrate, the direct-developing frog Eleutherodactylus coqui.

Mechanisms that mediate limb development are regarded as highly conserved among vertebrates, especially tetrapods. Yet, this assumption is based on the study of relatively few species, and virtually none of those that display any of a large number of specialized life-history or reproductive modes, which might be expected to affect developmental pattern or process. Direct development is an alternative life history found in many anuran amphibians. Many adult features that form after hatching in metamorphic frogs, such as limbs, appear during embryogenesis in direct-developing species. Limb development in the direct-developing frog Eleutherodactylus coqui presents a mosaic of apparently conserved and novel features. The former include the basic sequence and pattern of limb chondrogenesis, which are typical of anurans generally and appear largely unaffected by the gross shift in developmental timing; expression of Distal-less protein (Dlx) in the distal ectoderm; expression of the gene Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA); and the ability of the ZPA to induce supernumerary digits when transplanted to the anterior region of an early host limb bud. Novel features include the absence of a morphologically distinct apical ectodermal ridge, the ability of the limb to continue distal outgrowth and differentiation following removal of the distal ectoderm, and earlier cessation of the inductive ability of the ZPA. Attempts to represent tetrapod limb development as a developmental "module" must allow for this kind of evolutionary variation among species.     

Cranial neural-crest migration and chondrogenic fate in the oriental fire-bellied toad Bombina orientalis: Defining the ancestral pattern of head development in anuran amphibians

We assess cranial neural-crest cell migration and contributions to the larval chondrocranium in the phylogenetically basal and morphologically generalized anuran Bombina orientalis (Bombinatoridae). Methods used include microdissection, scanning electron microscopy, and vital dye labeling, in conjunction with confocal and fluorescence microscopy. Cranial neural-crest cells begin migrating before neural-fold closure and soon form three primary streams. These streams contribute to all cranial cartilages except two medial components of the hyobranchial skeleton (basihyal and basibranchial cartilages), the posterior portion of the trabecular plate, and the otic capsule, the embryonic origin of which is unknown. Chondrogenic fate is regionalized within the cranial neural folds, with the anterior regions contributing to anterior cartilages and the posterior regions to posterior cartilages. A neural-crest contribution also was consistently observed in several cranial nerves and the connective tissue component of many cranial muscles. Notwithstanding minor differences among species in the initial configuration of migratory streams, cranial neural-crest migration and chondrogenic potential in metamorphosing anurans seem to be highly stereotyped and evolutionarily conservative. This includes a primary role for the neural crest in the evolutionary origin of the paired suprarostral and infrarostral cartilages, two prominent caenogenetic features of the rostral skull unique to anuran larvae. Our results provide a model of the ancestral pattern of embryonic head development in anuran amphibians. This model can serve as a basis for examining the ontogenetic mechanisms that underlie the diversity of cranial morphology and development displayed by living frogs, as well as the evolutionary consequences of this diversity. © 1996 Wiley-Liss, Inc.     

Evolutionary innovation in the vertebrate jaw: A derived morphology in anuran tadpoles and its possible developmental origin

The mouthparts of anuran tadpoles are highly derived compared to those of caecilians or salamanders. The suprarostral cartilages support the tadpole's upper beak; the infrarostral cartilages support the lower beak. Both supra- and infrarostral cartilages are absent in other vertebrates. These differences reflect the evolutionary origin of a derived feeding mode in anuran tadpoles. We suggest that these unique cartilages stem from the evolution of new articulations within preexisting cartilages, rather than novel cartilage condensations. We propose testing this hypothesis through a search for similarities in the development of the suprarostral and infrarostral cartilage articulations and of the primary jaw joint. In Xenopus, the gene zax is expressed in a region corresponding to the infrarostral cartilage. This gene is related to the bapx1-gene, which regulates jaw joint development. Further investigation of these genes, as well as other genes with joint-related functions, in anuran craniofacial development may provide a connection between the morphological diversity seen in the vertebrate head and the corresponding diversity in genetic regulatory processes. We believe that the evolution of larval jaws in anurans may shed light on the general evolutionary mechanisms of how new articulations, not only in the jaw region, could have arisen in the vertebrate skull.     

A large‐scale survey of heterochrony in anuran cranial ossification patterns

Most anurans have a biphasic life cycle, which includes metamorphosis from a tadpole stage to an adult frog. This process involves extensive transformations of the cranial skeleton, which have been of long‐standing interest with respect to anuran skeletal evolution and taxonomy. In this study, large‐scale patterns of anuran skeletal ossification are assessed by collecting the most comprehensive data set on anuran cranial ossification to date from the literature, including data for 45 anuran and one caudate outgroup species. Ossification sequences were translated into event‐pair matrices for explorative phylogenetic analysis and phylogenetically informed parsimony search for heterochrony using the Parsimov algorithm. Rank variability of single bones across species was also analysed. Little phylogenetic signal was retrieved from a parsimony‐based phylogenetic analysis of event‐pairs, and only a few species that are generally agreed to be closely related are placed close to each other (e.g. some Pipidae and Costata). Parsimov analysis revealed some clade‐specific heterochrony in anuran clades of varying inclusiveness. Our results show that relating heterochronic changes in anuran cranial ontogeny to parameters such as direct development or miniaturization is problematic because of the high evolvability of cranial ossification sequences. Rank variation analysis suggests that anuran cranial bones are highly variable in their sequence positioning, possibly because tadpole and adult cranial morphology do not co‐evolve. Elements which are lost in some species ossify at the end of the sequence, providing evidence for the notion that failure of anuran cranial elements to ossify is due to processes of paedomorphosis.     

Cartilage on the move: Cartilage lineage tracing during tadpole metamorphosis

The reorganization of cranial cartilages during tadpole metamorphosis is a set of complex processes. The fates of larval cartilage‐forming cells (chondrocytes) and sources of adult chondrocytes are largely unknown. Individual larval cranial cartilages may either degenerate or remodel, while many adult cartilages appear to form de novo during metamorphosis. Determining the extent to which adult chondrocytes/cartilages are derived from larval chondrocytes during metamorphosis requires new techniques in chondrocyte lineage tracing. We have developed two transgenic systems to label cartilage cells throughout the body with fluorescent proteins. One system strongly labels early tadpole cartilages only. The other system inducibly labels forming cartilages at any developmental stage. We examined cartilages of the skull (viscero‐ and neurocranium), and identified larval cartilages that either resorb or remodel into adult cartilages. Our data show that the adult otic capsules, tecti anterius and posterius, hyale, and portions of Meckel's cartilage are derived from larval chondrocytes. Our data also suggest that most adult cartilages form de novo, though we cannot rule out the potential for extreme larval chondrocyte proliferation or de‐ and re‐differentiation, which could dilute our fluorescent protein signal. The transgenic lineage tracing strategies developed here are the first examples of inducible, skeleton‐specific, lineage tracing in Xenopus.     

DO LARVAL TRAITS RE‐EVOLVE? EVIDENCE FROM THE EMBRYOGENESIS OF A DIRECT‐DEVELOPING SALAMANDER,PLETHODON CINEREUS

Recent molecular phylogenies suggest the surprising reacquisition of posthatching metamorphosis within an otherwise direct-developing clade of lungless salamanders (family Plethodontidae). Metamorphosis was long regarded as plesiomorphic for plethodontids, yet the genus Desmognathus, which primarily includes metamorphosing species, is now nested within a much larger clade of direct-developing species. The extent to which the putative reacquisition of metamorphosis in Desmognathus represents a true evolutionary reversal is contingent upon the extent to which both larva-specific features and metamorphosis were actually lost during the evolution of direct development. In this study we analyze development of the hyobranchial skeleton, which is dramatically remodeled during salamander metamorphosis, in the direct-developing red-backed salamander, Plethodon cinereus. We find dramatic remodeling of the hyobranchial skeleton during embryogenesis in P. cinereus and the transient appearance of larva-specific cartilages. Hyobranchial development in this direct-developing plethodontid is highly similar to that in metamorphosing plethodontids (e.g., Desmognathus). The proposed reacquisition of hyobranchial metamorphosis within Desmognathus does not represent the "re-evolution" of a lost phenotype, but instead the elaboration of an existing developmental sequence.     

Developmental origins and evolution of jaws: new interpretation of "maxillary" and "mandibular"

Cartilage of the vertebrate jaw is derived from cranial neural crest cells that migrate to the first pharyngeal arch and form a dorsal "maxillary" and a ventral "mandibular" condensation. It has been assumed that the former gives rise to palatoquadrate and the latter to Meckel's (mandibular) cartilage. In anamniotes, these condensations were thought to form the framework for the bones of the adult jaw and, in amniotes, appear to prefigure the maxillary and mandibular facial prominences. Here, we directly test the contributions of these neural crest condensations in axolotl and chick embryos, as representatives of anamniote and amniote vertebrate groups, using molecular and morphological markers in combination with vital dye labeling of late-migrating cranial neural crest cells. Surprisingly, we find that both palatoquadrate and Meckel's cartilage derive solely from the ventral "mandibular" condensation. In contrast, the dorsal "maxillary" condensation contributes to trabecular cartilage of the neurocranium and forms part of the frontonasal process but does not contribute to jaw joints as previously assumed. These studies reveal the morphogenetic processes by which cranial neural crest cells within the first arch build the primordia for jaw cartilages and anterior cranium.