Extreme tadpoles II: the highly derived larval anatomy of Occidozyga baluensis (Boulenger, 1896), an obligate carnivorous tadpole

The fossil record of modern amphibians (frogs, salamanders, and caecilians) provides no evidence for major extinction or radiation episodes throughout most of the Mesozoic and early Tertiary. However, long-term gradual diversification is difficult to reconcile with the sensitivity of present-day amphibian faunas to rapid ecological changes and the incidence of similar environmental perturbations in the past that have been associated with high turnover rates in other land vertebrates. To provide a comprehensive overview of the history of amphibian diversification, we constructed a phylogenetic timetree based on a multigene data set of 3.75 kb for 171 species. Our analyses reveal several episodes of accelerated amphibian diversification, which do not fit models of gradual lineage accumulation. Global turning points in the phylogenetic and ecological diversification occurred after the end-Permian mass extinction and in the late Cretaceous. Fluctuations in amphibian diversification show strong temporal correlation with turnover rates in amniotes and the rise of angiosperm-dominated forests. Approximately 86% of modern frog species and >81% of salamander species descended from only five ancestral lineages that produced major radiations in the late Cretaceous and early Tertiary. This proportionally late accumulation of extant lineage diversity contrasts with the long evolutionary history of amphibians but is in line with the Tertiary increase in fossil abundance toward the present.

Preparation of small vertebrates cleared after alcian blue staining of cartilage is facilitated by trypsin digestion. Specimens are fixed in formation, washed, skinned, and eviscerated. After staining in a solution of alcian blue in acetic acid-alcohol for 24-48 hours, they are transferred to water through graded alcohols. Excess alcian blue is removed over a period of up to three weeks by changes every 2-3 days of 1% trypsin in approximately one-third-saturated sodium borate. Bony tissues may be stained after this in a solution of alizarin red S in 0.5% KOH. Specimens are bleached if necessary and dehydrated through graded KOH-glycerine mixtures for storage in glycerine. Since alcohol treatment in addition to formalin fixation does not affect results with this method, it should be useful to researchers who want to study the cartilage or cartilaginous skeletons in museum specimens, which are routinely fixed in formalin and stored in alcohol.