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The recent colonization of South America (Brncic and Budnik 1980; Brncic et al. 198 1) and North America (Beckenbach and Prevosti 1986; Prevosti et al. 1987) by the Paleartic species Drosophila subobscura has offered the opportunity to test empirically the effect of a founder event on different levels of polymorphism in an expanding population. Analysis of chromosomal inversion polymorphism (Prevosti et al. 1988), allozyme polymorphism (Prevosti et al. 1983), gametic associations of allozymes and chromosomal gene arrangements (Prevosti et al. 1983)) and allelism of lethals (Mestres et al., accepted) showed that only a mild founder effect occurred at the beginning of the colonization, but not during its expansion. Owing to its mode of inheritance, the variability of the mitochondrial DNA (mtDNA) is more sensitive to bottlenecks in population size than are nuclear genes. For this reason, it seems an ideal tool to get additional information about the colonization process. Although Latorre et al.‘s ( 1986) study of mtDNA variation in D. subobscura includes several American strains, their survey was not carried out at an intrapopulation level. In the present study, mtDNA variation has been analyzed in a total of 105 D. subobscura isofemale lines originating from North America (Eureka, 11; Gilroy, 26), South America (Santiago, 29; Valdivia, 6), and Europe (Barcelona, 33 ) . mtDNA was prepared by following a shorter version of our previous protocol (Afonso et al. 1988). The method is based on two steps of selective isolation of covalently closed circular DNA molecules, previously used for the isolation of plasmid DNA from bacteria. The first step involves alkaline denaturation (Birnboim and Doly 1979), and the second involves an acid-phenol extraction (Zasloff et al. 1978). One hundred fifty adult flies were used for its preparation, which gives enough pure mtDNA for lo12 restriction analyses. The same number of third-instar larvae gives similar yields. Flies are ground very gently in 1.75 ml precooled solution I [ 10 mM Tris, 60 mM NaCl, 5% sucrose (w/v), 10 mM EDTA (ethylenediamine tetracetate) pH = 8.01. Then 1.75 ml solution II [ 300 mM Tris, 5% sucrose (w/v), 10 mM EDTA, 1.25% sodium dodecyl sulfate ( SDS), 0.8% diethylpyrocarbonate (w/v) , freshly mixed] was added, and the solution was mixed and incubated at 65°C for 30 min. The DNA was denatured by adding 300 ~1 NaOH 2 N, so as to obtain a pH of 12.5, and was kept on ice for 6 min. SDS-potassium acetate (KAc) DNA deproteinization was performed by adding 600 ~1 KAc 5 M, pH = 5.0, and the resulting solution was mixed well and incubated at -20°C for 8 min. After centrifugation at 13,000 rpm for 10 min, the DNA-containing supematant was transferred to a 15-ml centrifuge tube. The acidphenol extraction was carried out by adding 4 ml phenol saturated with Tris-HCl (pH = 7.5), and the resulting solution was mixed by shaking and then was kept on ice for 5 min. The aqueous phase (pH 6-6.5)) was collected, and the traces of phenol were eliminated with a chloroform extraction. Finally the DNA precipitation was performed by adding 2 vol absolute ethanol. All samples were assayed for 11 restriction endonucleases: nine of them (BamHI,

Colonization of America by Drosophila subobscura: effect of the founder event on the mitochondrial DNA polymorphism.