Uzzell, T., H. Hotz, G.-D. Guex, and P. Beerli. 1994. Albumin cDNA sequences of western Palearctic water frogs: evolutionary changes in frog albumins. II. International symposium of ecology and genetics of European water frogs. September 1994. Wroclaw, Poland.

When protein electrophoresis began to be used for studying genetic variation in natural populations in the 1960s, the large amount of allelic polymorphism regularly observed contrasted surprisingly with the expectation of a well-adapted wild allele and some rare disadvantaged mutants. Recent technical advances now make it possible to study genetic variation within and among populations at the DNA nucleotide level, providing a powerful tool for evolutionary genetics with maximal resolution, maximal information, and access to the whole genome. We began sequencing cDNA for serum albumin in species of western Palearctic water frogs as part of our analysis of the evolutionary scenario that initiated clonal reproduction of natural interspecies hybrids in this group. We chose albumin because of its wide phylogenetic use in microcomplement fixation immunology and the relative ease with which its cDNA can be isolated; its generally high electrophoretic polymorphism in these frogs suggests relatively rapid divergence. We obtained reverse-transcribed cDNA from polyadenylated RNAfrom liver of four taxa (Rana shqiperica, an unnamed Sicilian taxon related to Rana lessonae, Rana bedriagae, Rana epeirotica), cloned it, identified albumin-positive plaques by hybridization with cloned Rana catesbeiana albumin cDNA, and sequenced the longest inserts in both directions. The four species studied are very similar in their albumin sequences, both at the nucleotide and at the amino acid level, paired differences ranging from 1.02 to 2.17% for coding nucleotides, from 2.30 to 5.39% for AAs. Two albumin genes of the Sicilian taxon (one from near Palermo, the other obtained from a hybridogenetic hybrid, Rana ridibunda x Sicilian taxon, from near Catania) were identical in all 1826 nucleotides compared (1641 in the coding region, 185 in the 3 untranslated region). This similarity of two populations, separated by >150 km of mountainous landscape, is striking. It is consistent with the low amount of allelic polymorphism detected by electrophoresis for other protein loci in the Sicilian taxon. Severe population bottlenecks during the Pleistocene cold periods could explain such low genetic diversity. Replacement nucleotide substitutions outnumbered silent ones in all paired comparisons. This is reflected in the % AA differences consistently being more than 1.5 times higher than the % nucleotide differences. This finding is not compatible with the expected preponderance of silent nucleotide changes inrelatively early divergence stages, reflecting selective disadvantage of many AA changes. It is possible that an unusually high portion of albumin AA replacements are not counterselected. That transversions in most comparisons outnumber transitions (grand mean over coding nucleotides 56%, maximum likelihood estimate over all coding sequences 65%) suggests caution in using the standard assumption of transition preponderance for estimation of phylogeny. Our results suggest a lower amount of hidden polymorphism at this locus than might be expected from the high electrophoretic diversity; this is testable by sequencing albumin cDNAs of several individuals of a variable population. Whether the albumin data are generalizable to other protein genes such as those coding for enzymes remains to be tested.

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