The egg jelly of Discoglossus pictus contains sperm motility-activating activity, the molecular basis of which has not been studied. Discoglossus pictus sperm initiated motility immediately after immersion in egg-jelly extract, as well as after immersion in hyposmotic solution, which initiates sperm motility in the external fertilization of anuran amphibians. Sequential treatment of the D. pictus sperm with these two solutions revealed the predominant effect of hyposmolality in initiation of motility. The motility initiation induced by jelly extract was suppressed by a monoclonal antibody (mAb) that is specific for the 34 kDa sperm motility-initiating substance (SMIS) in the egg jelly of the newt, Cynops pyrrhogaster. Immunoblotting using the anti-SMIS mAb revealed several antigenic proteins that included major ones with sizes of 18- and 34-kDa in D. pictus jelly extract. Scanning electron microscopic observation revealed that granules of jelly matrix, in which SMIS localizes and which have a critical role in the internal fertilization of C. pyrrhogaster, were not observed near the surface of the D. pictus egg jelly. These results suggest that sperm motility-activating activity in egg jelly of D. pictus may be mediated by SMIS homologous proteins that act through a mechanism that is partially different from that of C. pyrrhogaster.

Sequences of a mtDNA gene, cytochrome b, were used to generate a phylogenetic hypothesis for the evolution of Iberian archaeobatrachian frogs of the genus Discoglossus. Results from the inferred phylogenetic branching pattern and the sequence divergence among species and populations support the following: (1) the two endemic species from Iberia, D. galganoi and D. jeanneae are sister taxa and are reciprocally monophyletic with respect to their mtDNA; (2) the geographic distribution of these two morphologically cryptic species differs considerably from previous expectations. D. galganoi is limited to the metamorphic granitic and shale substrates of western Spain and Portugal, while D. jeanneae extends over the limestone and gypsum substrates of most of eastern Spain from the Strait of Gibraltar to the Pyrenees; (3) sequence divergence between D. galganoi and D. jeanneae averages 8.6%, suggesting that the two lineages diverged before the Mio–Pliocene boundary. We suggest that these lineages were isolated by the opening of the Betic Strait and that isolation has been maintained until recently by the Guadalquivir River Basin; (4) all specimens of D. jeanneae, from the central Pyrenees in the north to the southern end of the Iberian Peninsula have nearly identical cytochrome b haplotypes, clear evidence of a rapid relatively recent (Late Pleistocene–Quaternary) expansion of the species; (5) populations of D. pictus from northeastern Spain are highly divergent from all other Discoglossus studied and do not fall within the Iberian clade.