Sequence Of Cranial Ossification Research Articles

Intra- and interspecific variability of the cranial ossification sequences in Barbus sensu lato.

Intra- and interspecific variability of the cranial ossification sequences in Barbus sensu lato.

Skull development in the Iberian newt, Pleurodeles waltl (Salamandridae: Caudata: Amphibia): timing, sequence, variations, and thyroid hormone mediation of bone appearance

The cranial ossification sequence in Pleurodeles waltl is widely used in phylogenetic analyses of amphibian origin and evolution. However, the patterns published to date are far from completely resolved and contain certain discrepancies. Based on a large sample of P.waltl specimens ranging from early post-hatching larvae to post-metamorphic newts, we determined the most common cranial ossification sequence and revealed its intraspecific variations. Since thyroid hormones (THs) are involved in the mediation of skull development in salamanders, we studied the role of THs in the cranial development of P.waltl. The normal sequence and timing of bone appearance were compared with those in larvae reared under conditions of high (in 1 and 2ng mL-1 triiodothyronine) and low [in 0.02% thiourea (TU), which inhibits thyroid gland activity] TH levels. Metamorphosis was greatly accelerated in the TH-treated larvae and was arrested in the TU-treated larvae, which retained the larval pattern of the palate and rudimentary external gills even after 2 years of the experiment. Early-appearing bones (the coronoid, vomer, palatine, dentary, squamosal, premaxilla, parasphenoid, pterygoid, prearticular, vomer, frontal, parietal, exoccipital, in this order) arise at the same stages and ages, and follow the same ossification sequence under different TH levels. The timing of the appearance of bones normally arising in the late larval and metamorphic periods (the quadratojugal, orbitosphenoid, prootic, maxilla, nasal, os thyroideum, prefrontal, quadrate, in this order) changes depending on the TH level. The maxilla and nasal display the most pronounced reaction to changes in the TH level: they appear precociously in TH-treated animals, while their appearance is postponed and they remain rudimentary in TU-treated animals. Because of different responses to THs, the order in which late-arising bones appear changes depending on the TH level. Although bones appearing early in larval ontogeny (e.g. the premaxilla, vomer, squamosal, palatine) display no TH-induced reaction when they start to develop, their further differentiation shows dependence on THs, and these bones become TH-inducible closer to metamorphosis. These findings indicate that TH involvement in the mediation of cranial development changes from minimal (if at all) in its early stages to maximal during metamorphosis. It is likely that the appearance of bones early in development is mediated by factors other than THs. Their further development is accompanied by changes in the mechanisms mediating their morphological differentiation. That is, likely non-hormonal mediation becomes replaced or/and complemented by hormonal mediation. The constituent parts of the same bone may exhibit differences in their reactions to changes in TH levels. Although in normal development, the overall cranial ossification sequence is constant, there was variation in the order in which late-appearing bones was recorded. These observations suggest that this variation results from individual variability in the internal TH level. Comparison with other salamanders suggests that (a) the pattern of TH mediation described in P.waltl is common for cranial development of metamorphosing urodeles and (b) the same bone may differ in its TH dependence in different salamanders, e.g. there are interspecific variations in the degree of TH dependence of individual cranial bones.

Conserved in-ovo cranial ossification sequences of extant saurians allow estimation of embryonic dinosaur developmental stages

Dinosaur embryos are among the rarest of fossils, yet they provide a unique window into the palaeobiology of these animals. Estimating the developmental stage of dinosaur embryos is hindered by the lack of a quantitative method for age determination, by the scarcity of material, and by the difficulty in visualizing that material. Here we present the results of a broad inquiry, using 3D reconstructions from X-ray computed tomography data, into cranial ossification sequences in extant saurian taxa and in well-preserved embryos of the early branching sauropodomorph dinosaur Massospondylus carinatus. Our findings support deep-time conservation of cranial ossification sequences in saurians including dinosaurs, allowing us to develop a new method for estimating the relative developmental percentage of embryos from that clade. We also observe null-generation teeth in the Massospondylus carinatus embryos which get resorbed or shed before hatching, similar to those of geckos. These lines of evidence allow us to confidently estimate that the Massospondylus carinatus embryos are only approximately 60% through their incubation period, much younger than previously hypothesized. The overall consistency of our results with those of living saurians indicates that they can be generalized to other extinct members of that lineage, and therefore our method provides an independent means of assessing the developmental stage of extinct, in-ovo saurians.

Ossification of Atlantic cod (Gadus morhua) – Developmental stages revisited

In studies of marine larvae, it is common to use days post-hatch as a developmental reference point. We show that age is a poor measure of morphological and physiological development in Atlantic cod. Therefore, we propose a set of five developmental stages of Atlantic cod from start-feeding until the juvenile stage, based on cranial ossification as previously done in Atlantic halibut. Cod follows a sequence of cranial ossification that is to a large extent preserved in most fish species examined. These stages are therefore tools to standardize sampling and to reduce growth dependent variation in the analysis of larvae during development. We show that several developmental stages are present in the same rearing unit at a given time. We also demonstrate that nutrition during early development is a vital foundation for robust skeletal development. Cod larvae supplied with copepods instead of rotifers followed by Artemia, develop less skeletal deformities at 10cm standard length, despite given the same formulated feed from 1.8cm standard length and onwards. Statement of relevanceThis paper provides developmental stages that are vital for best practice protocols in aquaculture. By relating farming practices to developmental stages and not age, the right treatment ect may be provided.This manuscript does also highlight the importance of nutrition during live feed stages on events that may occur late in the production cycle.

Assessment of modularity in the urodele skull: an exploratory analysis using ossification sequence data.

The potential presence of developmental modules is studied in the urodele skull using several classical statistical methods that have not previously been used in this context. Principal component analysis (PCA) of ossification sequence data on 21 bones in 21 extant urodele species suggests the presence of up to four developmental modules, but examination of statistically significant correlations using phylogenetic independent contrasts (PIC) and correcting for multiple tests using the false discovery rate suggests the presence of only two modules of uneven size and of two bones that may not be part of these modules. Thus, PCA does not appear to be a reliable method to investigate modularity; direct investigation of statistically significant correlations using PIC or other phylogeny-informed methods is recommended. A binomial test of the distribution of significant correlations between characters shows significant heterogeneity, which suggests that modularity is indeed present in the data. A cluster analysis gives inconsistent results that apparently do not reflect developmental modules. The data include a phylogenetic signal, as shown by a permutation-based test with squared change parsimony, but this is detectable only when the whole matrix is analyzed, and a plot of the tree onto developmental space through Evolutionary PCA shows that homoplasy is pervasive. Evolutionary rates between characters vary about 90-fold. Canonical variates analyses suggest that obligatorily neotenic urodeles may be discriminated from other urodeles on the basis of cranial ossification sequence data.

Heterochrony and developmental modularity of cranial osteogenesis in lipotyphlan mammals.

BackgroundHere we provide the most comprehensive study to date on the cranial ossification sequence in Lipotyphla, the group which includes shrews, moles and hedgehogs. This unique group, which encapsulates diverse ecological modes, such as terrestrial, subterranean, and aquatic lifestyles, is used to examine the evolutionary lability of cranial osteogenesis and to investigate the modularity of development.ResultsAn acceleration of developmental timing of the vomeronasal complex has occurred in the common ancestor of moles. However, ossification of the nasal bone has shifted late in the more terrestrial shrew mole. Among the lipotyphlans, sequence heterochrony shows no significant association with modules derived from developmental origins (that is, neural crest cells vs. mesoderm derived parts) or with those derived from ossification modes (that is, dermal vs. endochondral ossification).ConclusionsThe drastic acceleration of vomeronasal development in moles is most likely coupled with the increased importance of the rostrum for digging and its use as a specialized tactile surface, both fossorial adaptations. The late development of the nasal in shrew moles, a condition also displayed by hedgehogs and shrews, is suggested to be the result of an ecological reversal to terrestrial lifestyle and reduced functional importance of the rostrum. As an overall pattern in lipotyphlans, our results reject the hypothesis that ossification sequence heterochrony occurs in modular fashion when considering the developmental patterns of the skull. We suggest that shifts in the cranial ossification sequence are not evolutionarily constrained by developmental origins or mode of ossification.

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. Die meisten Anura (Froschlurche) zeichnen sich durch einen biphasischen Lebenszyklus aus, während dessen sie eine Metamorphose vom Kaulquappenstadium zum adulten Frosch durchlaufen. Die Metamorphose geht mit weitreichenden Umformungen im kraniellen Skelett einher. Dieses Phänomen stößt seit langem im Rahmen von Fragestellungen mit Bezug auf Skelettevolution und taxonomische Klassifizierung auf Interesse. In der vorliegenden Studie werden anhand der bisher umfassendsten Datenerhebung, basierend auf der Literatur der letzten 50 Jahre, die Reihenfolge der Verknöcherung im Kranialskelett der Anura untersucht. Einbezogen wurden kranielle Verknöcherungs-sequenzen von 45 Vertretern der Anura und einem Vertreter der Caudata (Schwanzlurche). Die Sequenzen wurden als ‘Event-pairs’ kodiert, um eine informelle phylogenetische Analyse nach dem Sparsamkeitsprinzip sowie phylogenetisch basierte Heterochronieanalysen unter Anwendung des Parsimov-Algorithmus durchzuführen. Außerdem wurde die zwischenartliche Variabilität des Verknöcherungszeitpunktes innerhalb der Sequenz für die einzelnen Knochen untersucht. Die phylogenetische Analyse gruppierte einige Arten, welche generell als nahverwandt angesehen werden, z.B. einige Vertreter der Pipiden und Costata. Die Parsimov-Analyse zeigte, daß sich nur wenige Anurengruppen unterschiedlicher Diversität durch Heterochronien ausweisen. Unsere Ergebnisse zeigen, dass eine kausale Verbindung von Heterochronien mit Eigenschaften wie Direktentwicklung problematisch ist. Die Analyse der Verknöcherungsvariabilität weist auf große Variabilität des Verknöcherungs-Zeitpunkt der einzelnen Knochen hin; dies kann möglicherweise durch die relativ unabhängige Evolution von Larval- und Adultschädelmorhpologie erklärt werden. Elemente, die in einigen Arten nicht vorkommen, verknöchern generell am Ende der Sequenz. Dies spricht für die Annahme, daß der Verlust von Schädelknochen bei Anuren auf Pädomorphose zurückzuführen ist.

Looking closer at the determining of a phenotype? Compare by stages or size, not age

Summary During ontogeny, individual fish larvae and juveniles will develop at different paces despite apparently identical age and environment, and even genetic background. The urgent need to elucidate mechanisms critical to juvenile production, recruitment or even lifelong growth patterns implies that measures and results are compared by a meaningful standard such as developmental stage of the individual fish. Characterizations of developmental stages based on relatively invariable characteristics such as the sequence of cranial ossification have proven to be not only commercially and scientifically applicable in Atlantic halibut, but also point to a practical use for a character which is preserved in most fishes examined. Once described, useful proxies for stage determinants may be size parameters such as standard length, or preferably myotome height (particularly for flatfishes). The worst proxy for development of a batch of fish is age, exacerbated by the expense of analyzing irrelevant intervals.

Intraspecific Variation in Cranial Ossification in the Tailed Frog, Ascaphus truei

We describe the sequence of cranial ossification in the Tailed Frog, Ascaphus truei, and examine intraspecific variation in the timing and sequence of cranial development. Because tadpoles of A. truei lack the subarticular tubercles that normally delineate Gosner stages 39 and 40, we provide a modified Gosner staging system more appropriate for this species. Ossification sequence was highly variable among the populations and greater than that reported for other anuran species. In general, early ossification events in A. truei are similar to that reported for other anuran species. However, compared with other taxa, both onset of initial ossification events and appearance of specific elements appear relatively later in the development of Ascaphus.

AN EXPERIMENTAL STUDY OF INTRASPECIFIC VARIATION, DEVELOPMENTAL TIMING, AND HETEROCHRONY IN FISHES

Heterochrony is widely regarded as an important evolutionary mechanism, one that may underlie most, if not all, morphological evolution, yet relatively few studies have examined variation in the sequence of development. Even fewer studies have been designed so that intraspecific variation in the relative sequence of developmental events can be assessed, although this variation must be the basis for evolutionary change. Intraspecific variation in developmental ossification sequences was documented from the zebrafish (Danio rerio) by Cubbage and Mabee (1996) and from the Siamese fighting fish (Betta splendens) by Mabee and Trendler (1996), but a quantitative analysis of the patterns within this variation was not made. Here, we quantify the effect of rearing temperature on the sequence of ossification and characterize the levels and patterns of intraspecific variation in these fishes. For Danio, there were no temperature effects on the sequence of bone development across the cranium, cranial region development, cartilage versus dermal bones, or lateral line bone versus nonassociated bones. Likewise the level of variation in relative sequence (position) of ossification was low, about two ranks, across temperatures. At higher temperatures, we found higher levels of variation in iterated cranial bones and less in bones forming early in the sequence. No temperature effects on variation were found among regions, between lateral line–associated bones and nonassociated bones, between median and paired bones, or across the entire sequence, indicating concordant variability among the three temperatures. Individual bones with the highest levels of variability were not consistent among temperatures.Baseline patterns of intraspecific variation in Danio were compared to those of Betta. For both species, the level of intraspecific variation in sequence position was low and the variability of cranial bones was concordant. Individual bones with the highest levels of variability were not consistent between species. In both species, variation was widespread (distributed evenly across the sequence). We used comparisons (among regions, between dermal and cartilage bones, between lateral line–associated and other bones, between median and paired bones, between iterated and noniterated bones, between feeding-associated bones and others) to see which subsets were most variable and thus potentially useful in predicting high levels of evolutionary change. The only subset of bones that was significantly more variable than others was cartilage bones. If interspecific patterns are parallel to these intraspecific differences, cartilage bones would be expected to show higher levels of heterochrony. Although concordance across the cranial ossification sequence and among regions in Danio, Betta, and two other teleosts, Oryzias and Barbus, suggests an evolutionarily conserved pattern of ossification, identity in sequence position across taxa was not observed for any bone. Thus, variation existed in sequence position across temperatures and species. Intraspecific variation of this sort may influence the morphological outcome and evolutionary trajectories of species.Editor: M. Zeldich

Description of the chondrocranium and osteogenesis of the chacoan burrowing frog,Chacophrys pierotti (Anura: Leptodactylidae)

The larval chondrocranium of the large-headed leptodactylid frog, Chacophrys pierotti (Ceratophryinae), is described in detail. Descriptions include the ontogeny of the chondrocranium and osteogenesis of the cranial skeleton. The chondrocranium of C. pierotti is profoundly different from the chondrocrania previously described for the other genera of the Ceratophryinae (Ceratophrys and Lepidobatrachus). The chondrocranium of Chacophrys is longer than wide and not particularly robust or laterally expanded; that of Ceratophrys is very robust, whereas the chondrocranium of Lepidobatrachus is widely expanded laterally. These differences are particularly apparent in the elements associated with the jaw (i.e., suprarostral, infrarostral, Meckel's cartilage, palatoquadrate, cornua trabeculae), which are robust in Ceratophrys and thin and elongate in Lepidobatrachus. Unlike Ceratophrys and Lepidobatrachus, which possess highly specialized carnivorous larva, the chondrocranium of Chacophrys more closely resembles the typical microphagous herbivore morphology characteristic of other leptodactylid frogs for which the chondrocrania are known. These data suggest that Chacophrys is the basal taxon within the monophyletic Ceratophryinae. The ontogeny of the chondrocranium of Chacophrys, as well as the cranial ossification sequence, do not differ greatly from those described for Ceratophrys. Detailed descriptions of the ontogeny of the chondrocranium and the bony skeleton are needed for additional taxa within the Ceratophryinae (especially Lepidobatrachus). Such descriptive ontogenetic studies promise new insight into the phylogeny and morphological evolution of this remarkable group of large-headed frogs.

The Chondrocranium of the Mexican Burrowing Toad, Rhinophrynus dorsalis

-The chondrocranium of Rhinophrynus dorsalis is described and illustrated. Autapomorphies of R. dorsalis are the presence of the symplectic cartilage and a cartilaginous process on the ascending process of the palatoquadrate. Synapomorphies of Rhinophrynus shared with the Pipidae are: (1) early formation of the lower jaw and (2) presence of a suprarostral plate. Alternative developmental pathways for the formation of the suprarostral plate are considered. Histological data indicates that the suprarostral plate forms from: (1) the early fusion of the cornua trabeculae with the suprarostral cartilages, (2) an anterior growth of the ethmoid plate, and (3) subsequent fusion of the cornua trabeculae and the anterior process of the ethmoid plate. The larval crista parotica of R. dorsalis resembles the one described for microhylid taxa. Taylor (1942) described pipid-like anuran larvae from a sample of tadpoles collected in Guerrero, Mexico. However, these larvae remained unidentified until the tadpole of Rhinophrynus dorsalis was described by Orton (1943). Orton's description included a few chondrocranial characteristics, particularly focusing on the jaw apparatus. She noted the early formation of the lower jaw and suggested that this early development may be correlated with a macrophagous diet. Subsequently, Sokol (1977, 1981) made references to the chondrocranial anatomy for Rhinophrynus dorsalis larvae while evaluating the use of larval characteristics in systematics. The sequence of cranial ossification for Rhinophrynus larvae was described by Trueb, 1985. Description of adult osteology has been previously described (Trueb, 1973; Trueb and Cannatella, 1982). Hay et al. (1995) placed Rhinophrynus as the sister group to Pipidae based on 12S and 16S rRNA sequence data. Previous morphological studies suggested the following synapomorphies for the Pipoidea: absence of mentomeckelian bones, lack of parasphenoid alae, presence of a single frontoparietal, greatly enlarged otic capsules, and larvae with paired spiracles and lacking comified beaks and denticles (Ford and Cannatella, 1993). Of the approximately 28 species of the Pipoidea, the chondrocranium has been described thoroughly for Xenopus laevis (Trueb and Hanken, 1992) and partially for Rhinophrynus dorsalis, Pipa carvalhoi, and Xenopus tropicalis (Sokol, 1975). Herein we provide a complete description and illustration of the chondrocranium of Rhinophrynus dorsalis. 1 Corresponding Author. MATERIALS AND METHODS Thirty tadpoles of the Mexican Burrowing Toad, Rhinophrynus dorsalis, representing Neiuwkoop and Faber (1956) stages 48-62 were cleared and double-stained for bone and cartilage using standard procedures. Drawings were obtained with the help of a Wild M3C stereomicroscope with camera lucida attachment. Collection localities of specimens used in this study are: Guanacaste, Costa Rica (USNM 515945515958); and Aguacate Lagoon, Cayo District, Belize (USNM 515959-515974). Collection numbers, measurements, and stages of material examined are given in Table 1. Nieuwkoop and Faber's (1956) staging table for Xenopus was used to stage Rhinophrynus tadpoles. The similarity of external morphology of Xenopus and Rhinophrynus during development makes this table preferable to the Gosner's (1960) staging table. Chondrocranial terminology follows De Jongh (1968), Haas (1995), and Trueb and Hanken (1992).

Development of the cranium and paired fins in Betta splendens (Teleostei: Percomorpha): Intraspecific variation and interspecific comparisons.

The development of the chondrocranium and the relative timing of ossification of the osteocranium is described in the teleost fish Betta splendens from a large series of cleared and differentially stained specimens. General trends in ossification patterns are examined from developmental, phylogenetic, and functional contexts. As in many other vertebrates, dermal bones form before cartilage bones. Ossification sequence conforms to functional need in a very general way, but there are many inconsistencies in the details of order. For example, some bones that are directly involved in feeding ossify no earlier than bones more indirectly involved. Comparisons of ossification sequence within specific cranial regions are made among Betta splendens, Oryzias latipes (Atherinomorpha), and Barbus barbus (Ostariophysi) within a phylogenetic framework. Many evolutionary changes in relative sequence of ossification are evident within regions among these taxa, yet many other sequences are conserved. The logistic difficulty of comparing entire cranial ossification sequences (vs. regional sequences) makes evident the need for new methods for identifying and quantifying sequence changes. Intraspecific variation in order of ossification is described for the first time in teleost fishes. To the extent that ossification sequence varies intraspecifically, conclusions drawn from previous interspecific comparisons are compromised. Understanding the importance of changes in ossification order within and among taxa will require experimental, functional, and evolutionary work. © 1996 Wiley-Liss, Inc.

The influence of variation in larval period on adult cranial diversity in Pelobates fuscus (Anura: Pelobatidae)

The timing and sequence of cranial ossification was studied in Pelobates fuscus tadpoles differing in the rate of their larval development. Laboratory‐reared animals, obtained from one egg mass, were examined. Some of the tadpoles metamorphosed during the first summer (normally developing tadpoles), whereas others prolonged the timing of their larval development into a second year or even later (slowly developing tadpoles). In slowly developing tadpoles the timing and sequence of cranial ossification differ in many respects from those occurring in normally developing tadpoles. Thus (a) ossification of some bones commences earlier and (b) there are some anachronisms in the sequence of ossification. As a whole, different cranial elements respond differently to prolongation of larval development. Bones which arise early in normal ontogeny (parasphenoid, frontoparietals, prootics, exoccipitals and interparietal) have a lengthened period of morphological differentiation and growth when larval development is prolonged. Consequently they show hypermorphosis in the adult with a profound effect on adult cranial morphology. Other ossifications, whose origin and growth are associated with the final metamorphic event (the transformation of the larval into the adult jaw mechanism), show no evident changes in the timing of their origin relative to somatic development.Another mechanism for altering adult morphology in animals with prolonged larval development results from the dissociation between somatic and sexual development. While somatic development in slowly developing tadpoles is very retarded and their external morphology may show no change during a large part of the larval stage, the generative system continues to develop. This situation produces tadpoles with premetamorphic somatic morphology but with almost mature generative organs. These tadpoles retain the ability to metamorphose and consequently they may transform into sexually mature animals with juvenile (early postmetamorphic) somatic morphology, i.e. into neotenic animals. In some specimens early‐originating cranial bones may be hypermorphosed whereas cranial elements normally arising in late metamorphic stages, or after metamorphosis is complete, are subject to paedomorphic reduction and under‐development.It is concluded that wide individual variation in the rate of larval development may account, at least in part, for the occurrence of intraspecific variability in anuran cranial morphology (e.g. in Pelobates syriacus).

Skull development during anuran metamorphosis. II. Role of thyroid hormone in osteogenesis.

We examined the role of thyroid hormone (TH) in mediating cranial ossification during metamorphosis in the Oriental fire-bellied toad, Bombina orientalis. Exogenous T3 (3,3',5-triiodo-L-thyronine) was administered in three treatment dosages (0.025, 0.25, and 2.5 micrograms) plus a control dosage via plastic micropellets implanted within the dermis of tadpoles of three Gosner developmental stages: 28/29, 30/31, 32/33. Tadpoles were recovered after 2, 4, 6, and 8 d, and scored for the presence of three bones - median parasphenoid and paired frontoparietals and exoccipitals--as seen in cleared-and-stained, whole-mount preparations. T3 induced precocious ossification in both a stage-dependent and a dosage-dependent manner; stage dependence corresponded precisely with the degree of osteogenic differentiation at the time of hormone administration. Precocious ossification thus was due to the T3-promoted growth and calcified matrix deposition of these centers. Differential TH sensitivity among osteogenic sites may underlie both the temporal cranial ossification sequences characteristic of metamorphosing amphibians as well as sequence differences commonly observed among taxa.

Chondrocranium and ossification sequence of Hyla lanciformis.

Tadpoles of Hyla lanciformis live in midwater and have a normal, beaked, anteroventral, subterminal mouth. Overall, they are generalized pond-type anuran larvae that show no specific modification for a particular habitat. The chondrocranium and ossification sequences of this hylid frog were studied, utilizing cleared and Alcian blue-alizarin red-stained tadpoles. The chondrocranium is similar to that of other unspecialized tadpoles. No larval otic process was found and a large process projecting from the otic capsule is considered homologous with the larval crista parotica described for other anurans. The pattern of cranial and postcranial ossification is reported. The sequence of cranial ossification is explained in the framework of functional changes of the skull through metamorphosis. Comparison with the available information for other hylid frogs shows clear differences in the ossification sequences. The sequence of postcranial ossification is described for the first time for any species of hylid frog.

Variation and timing of the cranial ossification sequence of the oriental fire-bellied toad, Bombina orientalis (Amphibia, Discoglossidae).

The sequence of appearance of the 17 different skull bones in the oriental fire-bellied toad, Bombina orientalis, is described. Data are based primarily on samples of ten or 11 laboratory-reared specimens of each of 11 Gosner developmental stages (36-46) representing middle through late metamorphosis. Ossification commences as early as stage 37 (hind limb with all five toes distinct), but the full complement of adult bones is not attained until stage 46 (metamorphosis complete). Number of bones present at intermediate stages is poorly correlated with external morphology. As many as four Gosner developmental stages elapse before a given bone is present in all specimens following the stage at which it may first appear. The modal ossification sequence is frontoparietal, exoccipital, parasphenoid, septomaxilla, premaxilla, vomer, nasal, maxilla, angulosplenial, dentary, squamosal, quadratojugal, pterygoid, prootic, interfrontal, sphenethmoid, and mentomeckelian. Most specimens are consistent with this sequence, despite the poor correlation between cranial ossification and external development as assayed by Gosner stage. The timing of cranial ossification in Bombina orientalis differs in many respects from that described for two other, distantly related anurans, the leopard frog (Rana pipiens) and the western toad (Bufo boreas). These include the total number and sequence of appearance of bones, and the timing of ossification relative to the development of external morphology. Interspecific variation may reflect differences in the timing of the tissue interactions known to underlie skeletal differentiation and evolution.

The Sequence of Cranial Ossification in the California Toad, Bufo boreas (Amphibia, Anura, Bufonidae)

The order of appearance of skull bones in Bufo boreas is described. Skull bones appear in the following sequence: exoccipitals, parasphenoid, frontoparietals, premaxillae, prootics, squamosals, angulares, dentaries, septompxillae, maxillae, nasals, pterygoids, prevomers, palatines, quadratojugals, mentomeckelians, columellae, and sphenethmoid. Overall skull devel- opmental patterns in B. boreas did not differ greatly from those in other generalized frogs; however, differences were noted in the specific developmental patterns of the frontoparietal, exoccipital, columella, and sphenethmoid. Differences in overall developmental patterns were noted when comparisons were made with Xenopus laevis, Ascaphus truei, Osteopilus septentri- onalis, Smilisca baudinii, and Triprion petasatus. Comparisons among all of the species studied to date indicate several instances of fusion of, or loss of ossification centers in the skull of Bufo boreas. A comparison of osteogenesis in field-collected and lab-reared B. boreas revealed that even though growth patterns and sequence of appearance is the same in both groups, head bones develop significantly earlier in toads collected in the field. Head bones also developed earlier in toads collected in the Owens Valley of eastern California than in the San Fernando Valley of southern California. * * *