Freeze tolerance evolution among anurans: Frequency and timing of appearance

Despite numerous mechanistic studies on physiological responses supporting freeze tolerance in anurans, few have addressed the evolutionary significance of this trait. We thus investigated the phylogenetic relationships among anuran species whose freeze tolerance has been assessed and in combination with new data on freezing tolerance of two closely related species of the European brown frogs (Rana temporaria and Rana dalmatina). The species we studied exhibited short survival times in frozen state (around 8 h for both species). Phylogenetic analysis suggests that freeze tolerance evolved at least two times among Ranidae and one or two times among Hylidae and never in Bufonidae. Furthermore, in order to assess the timing of divergence of this character we used a relaxed molecular clock created, and found that the most recent separation between a freeze tolerant species and a freeze intolerant species dates from 15.9 +/- 7.6 Myr (Rana arvalis and R. temporaria). The comparison between these two species thus represents the best current model to understand freeze tolerance evolution. Addressing the evolution of this trait with such large-scale approaches will not only improve our understanding of cold hardiness strategies, but might also create a framework guiding future comparative studies.