We examined whether a remarkable occurrence -the physiological evolution of two Drosophila melanogaster populations, despite a spatial separation of only 100-400 m, was idiosyncratic and temporary, or persisted over multiple years. We ascertained the high-temperature tolerance of Drosophila descended from populations on the north-facing slope (NFS) and south-facing slope (SFS) of 'Evolution Canyon' (Lower Nahal Oren, Mt Carmel, Israel), which were collected in 1997, 1999, and 2000. Results for these Drosophila uniformly resembled other studies in many respects: an inverse relationship between survival and heat-shock temperature, male-female differences in thermotolerance, and inducible thermotolerance. Importantly, for all years of collection, SFS flies consistently exceeded NFS flies in basal and inducible thermotolerance after diverse heat shocks, with and without thermal pretreatment, and whether isofemale lines, synthetic populations, or inbred lines were compared. Inbred lines, however, had lower thermotolerance than outbred lines. Several nonexclusive processes may explain the evolution of such physiological differentiation.

The repair efficiency of four thermotolerant and four thermosensitive isofemale lines of Drosophila melanogaster originating from "Evolution Canyon" (Mt Carmel, Israel) was tested using 2-acetylaminofluorene (2-AAF) as mutagen. First, males of the standard laboratory line Canton S were treated with either 2-AAF solution or control solution. Then, females of the "Evolution Canyon" lines were crossed with treated (2-AAF or control solution) males and maintained at either 24 or 29 degrees C. Arbitrary primed PCR fingerprinting was employed as a method for genomic damage analysis in the resulting progeny (by scoring the frequency of lost DNA bands in F(1) progeny). Thermosensitive lines displayed significantly higher rates of change in the DNA fingerprint pattern after mutagenic presyngamic treatment followed by development at both temperatures, as well as after development under high temperature with no prior mutagenic treatment. The thermotolerant lines tended to show a lower level of mutation at both temperatures and after both treatments. One isofemale line showed a higher level of mutation at room temperature compared with increased temperature, after both control and mutagen treatment. The results suggest the existence of a relationship between DNA repair efficiency and thermotolerance, with thermotolerant lines tending to repair DNA more efficiently than thermosensitive ones.

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