GEOLOGICALLY DATED SEA BARRIERS CALIBRATE A PROTEIN CLOCK FOR AEGEAN WATER FROGS

Reliable estimates of phylogenetic relationships and divergence times are a crucial requirement for many evolutionary studies, but are usual ly difficult because fossils are scarce and their interpretation is of ten uncertain. Frogs are fresh water animals that generally are unable to cross salt water barriers (their skin is readily permeable to prov ide an independent measure of the minimum date of genetic divergence b etween pairs of such populations. For the genetically well-studied wes tern Palearctic water frogs (Rana esculenta group), the Aegean region provides an ideal area for determining the relationship between geneti c divergence and time of spatial isolation, using a nested set of geol ogically determined isolation times (12,000 yr, 200,000 yr, 1.8 Myr, 2 -3 Myr, and 5.2 Myr). Using 31 electrophoretic loci for 33 pairs of ne ighboring frog populations, a linear relationship between geologically determined isolation time and Hillis' modified Nei genetic distance w as found: D-Nei = (0.04 +/- 0.01) + (0.10 +/- 0.01) isolation time [M yr] corresponding to an average divergence rate (''molecular clock'' p ace) of 0.10 D-Nei/Myr (0.10 D-Nei/Myr). This rate is in the range of previous estimates reported for protein electrophoretic data; the val ue is conservative because relatively few of the loci used are ''fast evolvers'' (13%; sAAT, ALE, EST-5, MPI). Removing these fast evolvers from the analysis results in 0.08 D-Nei/Myr (0.08 D-Nei/Myr). The con fidence limits for estimation of the divergence time given the genetic distance are large, but unusually narrow for this kind of study; they permit us to estimate divergence times during the Pliocene and Miocen e. Few previous studies, including sequence analyses, have provided re asonable estimates of divergence time for the Pliocene. A test using t he outgroup taxa Rana perezi and Rana saharica (also isolated for 5.2 Myr by the Strait of Gibraltar) fits the calibration well: observed ge netic Nei distance D-Nei 0.55, expected D-Nei* 0.56. The calculated d ivergence times, based on this absolute molecular clock, suggest a ser ies of speciation events after the Messinian (5.2 Myr), possibly trigg ered by the rapid ecological changes accompanying the desiccation and refilling of the Mediterranean Basin.