The highest altitude recorded for an amphibian in Europe (west of the Caucasus) is 2965 m. It refers to the "lacs de Cambalès" according to an old reference. However, these lakes are all situated below 2600 m, while the altitude corresponds exactly to that of the summit of the Pic de Cambalès. We undertook an almost complete survey of ponds in a 2.5 km radius from the Cambalès peak, and complemented these data by fieldwork in a larger portion of the Central Pyrenees. Based on our observations in a total of 325 lakes and ponds we conclude that no water bodies above 2800 m exist in the Cambalès area, and that no water bodies above 2600 m are populated by Rana temporaria. The highest amphibian record was that of a single R. temporaria specimen at 2569 m, while the highest reproducing population was found at 2516 m. Highest records of other amphibians were 2516 m (Alytes obstetricans), 2160 m (Bufo bufo), 2259 m (Euproctus asper), and 2142 m (Salamandra salamandra). The presence of ice-free water bodies during a sufficient amount of time for larval development is probably the limiting factor for elevational distribution of Pyrenean amphibians, and the adequate conditions are usually not met above 2500 m in this massif. In contrast, in the Alps, ponds at higher altitudes are often protected by much higher surrounding peaks, which leads to higher elevational occurrence of amphibians. The Italian Laghi di Tre Becchi (up to 2742 m) are therefore the highest locality of a reproducing anuran population in Europe west of the Caucasus.
Genetic differentiation of Rana temporaria from the Pyrenean and Cantabrian mountains in Spain was studied by means of allozyme electrophoresis. 24 loci were analysed in 104 specimens from 15 populations: nine populations from the Pyrenean massif, five populations from the area of the Cantabrian mountain chain (regions of Galicia, Asturias, and Basque Country), and one population from Germany. Three distinct clusters were distinguished by phenetic analysis: (a) the Pyrenean samples and the single population from the Basque Country, (b) the populations from Galicia and Asturias) and (c) the German population. Ordination (PCA) resulted in one principle component (PC1) that separated Cantabrian from Pyrenean populations, and in a second one (PC2) that separated the single German population from the Iberian ones. PC1 indicated introgression that was corroborated by west-east clines in several alleles along the Cantabrian chain. The rather clear separation of the Cantabrian and Pyrenean clusters (mean genetic distance 0.121) suggests that two genetically different subspecies of R. temporaria may be distinguished in Spain. The absence of fixed allelic differences between populations refutes recent hypotheses of the existence of syntopic sibling species within R. temporaria in Spain. Biogeographically, the Pyrenean and Cantabrian populations possibly originated in two separate colonisation events starting from different glacial refuges. The strong morphological differentiation of Pyrenean R. temporaria populations is not paralleled by genetic divergence, and may better be explained by ecological factors such as climate, altitude and vegetation.