JOURNAL OF MORPHOLOGY 195:247-256 (1988) Skull Development During Anuran Metamorphosis: I. Early Development of the First Three Bones to Form-The Exocspital, the Parasphenoid, and the Frontoparietal JAMES HANKEN AND BRIAN K. HALL Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309-0334 (J.H.); Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H4Jl (B.K.H.) ABSTRACT In anuran amphibians, crania1 bones typically first form at metamorphosis when they rapidly invest or replace the cartilaginous larval skull. We describe early development of the first three bones to form in the Oriental fire-bellied toad, Bombina orientalis-the parasphenoid, the fronto- parietal, and the exoccipital-based on examination of serial sections. Each of these bones is fully differentiated by Gosner stage 31 (hindlimb in paddle stage) during premetamorphosis. This is at least six Gosner developmental stages before they are first visible in whole-mount preparations at the begin- ning of prometamorphosis. Thus, developmental events that precede and me- diate the initial differentiation of these cranial osteogenic sites occur very early in metamorphosis-a period generally considered to lack significant morphological change. Subsequent development of these centers at later stages primarily reflects cell proliferation and calcified matrix deposition, possibly in response to increased circulating levels of thyroid hormone which are charac- teristic of later metamorphic stages. Interspecific differences in the timing of cranial ossification may reflect one or both of these phases of bone develop- ment. These results may qualify the use of whole-mount preparations for inferring the sequence and absolute timing of cranial ossification in am- phibians. Amphibian metamorphosis, particularly in anurans, entails a tremendous morphologi- cal restructuring of virtually every organ system in the body. This restructuring is no- where more conspicuous than in the skull, where the cartilaginous larval cranium is rapidly converted into the primarily bony skull of the postmetamorphic froglet. Here, as is typical for osteocranial development in vertebrates generally, the bones form in a stereotyped, species-specificsequence that is correlated closely in time with other devel- opmental events. These broad features of cranial metamor- phosis have been known for some time, yet detailed knowledge of the development of the osteocranium in amphibians is available for surprisingly few taxa. Indeed, descriptions of the complete sequence and timing of cranial ossification are known for only 18 of the more than 3,400 extant species of anurans (Duell- man and Trueb, '86; Trueb, '85). Information is particularly sparse concerning the embry- onic and larval precursors of osteogenic cells and the nature and timing of the develop- mental processes that underlie their differ- entiation into discrete bony elements. In an earlier study, we described the se- quence and timing of cranial ossification in the Oriental fire-bellied toad, Bombina orien- talis, as a prelude to experimental investiga- tions of the developmental mechanisms that underlie cranial osteogenesis in this species (Hanken and Hall, '84). Based on examina- tion of cleared, whole-mount preparations differentially stained for bone and cartilage, we found that the first cranial bones to form are the parasphenoid and the paired fronto- parietals and exoccipitals-bones which were visible no earlier than Gosner stage 37 (pro- metamorphosis-Etkin, '35). Subsequently, we gathered additional descriptive data, pre- 0 1988 ALAN R. LISS, INC