Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2145-2149, June 1975 Genetic Variation As a Test of Natural Selection (allozymes/heterozygosity/environmental heterogeneity) EVIATAR NEVO*, HERBERT C. DESSAUERt, AND KUO-CHIIAN CHUANGt * Department of Biology, University of Haifa, Haifa, Israel; and t Department of Biochemistry, Louisiana State University Medical Center, New Orleans, La. 70112 Communicated by Theodosius Dobzhansky, February 6, 1976 ABSTRACT Allozymic variation encoded by 26 loci was 27,C); (7) Nafah (n = 24,C); (8) Jericho (n = 58,1); (9) analyzed electrophoretically in 54f7 specimens representing El-Arish (n = 46,1); (10) Mashabei-Sade (n 56,1); (11) 12 populations of green toads, Bufo viridis, in Israel and the Vis Adriatic Island. Genetic variation in Bufo viridis is Ein-Ovdat (n = 53,I); and (12) Vis Adriatic Island (n = higher than in any vertebrate yet studied. Mean hetero- 10,1). (Populations 1-11 are from Israel.) zygosity per locus per individual (H) is 0.133 (range, 0.105 The Israeli populations of Bufo viridis live under extreme to 0.159). H is higher in central populations as compared ecological conditions in both space and time. Spatially, they with isolates, and varies among four major protein classes, range along ecological gradients of increasing aridity south- being highest in transferases and hydrolases and lowest in and oxidoreductases and nonenzymatic proteins. Differential wards as well as eastwards, approaching the southern gene frequencies among polymorphisms was tested as an eastern deserts surrounding Israel. While annual precipitation indicator of natural selection. Significant heterogeneity in the northern populations of Quneitra, Nafah, Dalton, and between loci in their apparent inbreeding coefficients Saar is 850, 850, 700, and 650 mm, respectively, it decreases F. = 2p/p( -Tp) was found for all alleles and for each of to arid desert conditions in the southern populations of El- the four major classes of proteins tested, which may be taken as evidence of selection. Both uniform and diversify- Arish, Mashabei-Sade, and Ein-Ovdat, which receive annual ing selection are suggested by the low and high F, values, precipitations of 100, 100, 75, and 65 mm, respectively. respectively. The general pattern of high heterozygosity Temporally, all Israeli populations live either under Medi- in Bufo viridis is best explained as an adaptive strategy in terranean or desert climates involving mild wet winters but heterogeneous environments. dry hot summers. Average temperatures across the range of The pattern of genetic variation within and between popula- the sampled populations are for the entire year 19.10 (range, tions may be used to test directly the alternative theories of 16-24°); for the coldest month (January) 10.50 (range, the "balanced" versus the "neo-classical" or "neutral" 6-15°), and for the hottest month (August) 25.8° (range, schools of evolutionary genetics (1-4). While natural selec- 22-31°). Bufo tadpoles metamorphose in spring and early tion operates differentially on each allele, the breeding struc- summer and are thus exposed in Israel to adverse and dry ture, involving random genetic drift, inbreeding, assortative environments. Adult green toads are active during summer mating, and migration, affects all alleles similarly. Differential nights and walk long distances in search of food or mates in genic variations among polymorphisms in space and time the breeding season, thus being also exposed to environmental may thus provide evidence of selection in natural populations extremes (5). (3,4). Genetic variation may be expressed as the variance (S2) We present evidence based on differential gene frequencies of allele frequencies across populations. To overcome the of 12 populations of green toads, Bufo viridi8, in Israel and effect of the mean gene frequency (p) in computing the on the Vis Adriatic Island (H. C. Dessauer, E. Nevo, and variance, the latter may be standardized, as in the estimate K. C. Chuang, submitted for publication), suggesting that e= S2p/P (1 - P) known as the Wahlund variance (6), natural selection is the major operating evolutionary force or the "effective inbreeding coefficient" (7). (See also ref. 3 causing population differentiation. Furthermore, green toads for the statistical properties of Pe and for a sampling theory probably demonstrate an adaptive strategy for high hetero- for testing heterogeneity in ke.) Selectively neutral alleles zygosity in accord with their ecologically variable range in will all have similar estimated Pe values in spite of their space and time. variation in S2p and p because effective inbreeding will be identical for all genes across populations. In that neutral case, the average Pe will thus estimate the true Pe without MATERIALS AND METHODS significant heterogeneity among alleles. Lewontin and Kra- Allozymic variation encoded by 26 loci was analyzed electro- kauer (3) have showed that the theoretical variance for a phoretically in 507 adult specimens representing 12 popula- sampling distribution of Pe values from alleles not subject to tions (5 central, 2 marginal, and 5 isolates) of green toads, selection is approximately u2 = (KF2)/(n - 1) where F is Bufo viridie, 11 populations in Israel and 1 population in the the mean Fe value, n is the number of populations under Vis Adriatic Island. The 12 populations, sample sizes, and study, and K = 2, assuming a binomial distribution of allele their geographical location, central (C), marginal (M), and frequencies. If, however, selection is operating on some or all isolate (I), are as follows: (1) Saar (n = 73,C); (2) Tel-Aviv of the loci, their Pe values will be significantly heterogeneous (n = 28,C); (3) Ein Hashlosha (n = 53,M); (4) Dalton and will not be estimates of the same Pe. The ratio of the (n = 47,C); (5) Jerusalem (n = 42,M); (6) Quneitra (n = observed variance of Pe's (S2F) to the theoretical variance 2145 Downloaded by guest on September 27, 2021 2146 Genetics: Nevo et al. Proc. Nat. Acad. Sci. USA 72 (1975) TABLE 1. Gene frequencies and effective inbreeding coefficients of alleles at 20 polymorphic loci in populations of Bufo viridis* Ein Saar Tel- Hash- Jeru- El- Mashabei Ein- With- n = Aviv losha Dalton salem Quneitra Nafah Jericho Arish Sade Ovdat Vis out With Locust Allele (73)1t (28) (53) (47) (42) (27) (24) (58) (46) (56) (53) (10) Vis Vis I. O 1i9oreductases Ldh ba 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.991 1.000 1.000 1.000 1.000 0.009 0.009 b 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.009 0.000 0.000 0.000 0.000 0.009 0.009 Ldh-2 a 0.000 0.000 0.000 0.011 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.011 0.011 b 0.281 0.429 0.226 0.298 0.048 0.278 0.208 0.231 0.272 0.241 0.047 0.000 0.064 0.091 c 0.719 0.571 0.774 0.691 0.952 0.722 0.792 0.769 0.728 0.759 0.953 1.000 0.067 0.072 Mdh-i a 1.000 1.000 1.000 1.000 0.988 1.000 0.958 1.000 1.000 1.000 1.000 1.000 0.033 0.034 b 0.000 0.000 0.000 0.000 0.012 0.000 0.042 0.000 0.000 0.000 0.000 0.000 0.034 0.034 Icd-i a 0.058 0.019 0.028 0.021 0.115 0.065 0.043 0.070 0.000 0.018 0.000 0.000 0.032 0.035 b 0.507 0.685 0.519 0.426 0.500 0.435 0.391 0.480 0.598 0.670 0.702 0.650 0.048 0.048 c 0.435 0.296 0.453 0.553 0.385 0.500 0.565 0.450 0.402 0.313 0.298 0.350 0.037 0.036 Icdc2 a 0.021 0.036 0.009 0.000 0.000 0.038 0.000 0.000 0.000 0.009 0.000 0.000 0.021 0.022 b 0.979 0.928 0.991 1.000 1.000 0.962 1.000 1.000 1.000 0.991 1.000 1.000 0.039 0.040 c 0.000 0.036 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.036 0.036 6Pgd a 0.979 0.964 0.981 0.947 1.000 0.944 0.979 0.991 0.989 1.000 1.000 1.000 0.020 0.022 b 0.021 0.036 0.019 0.053 0.000 0.056 0.021 0.009 0.011 0.000 0.000 0.000 0.020 0.022 Ipo a 0.000 0.000 0.000 0.000 0.024 0.000 0.000 0.034 0.000 0.000 0.000 1.000 0.027 1.027 b 1.000 1.000 1.000 1.000 0.976 1.000 1.000 0.966 1.000 1.000 1.000 0.000 0.027 1.027 II. Transferases Got-i a 0.000 0.000 0.009 0.000 0.000 0.000 0.000 0.009 0.000 0.000 0.000 0.000 0.008 0.008 b 0.959 0.982 0.982 0.947 1.000 0.963 1.000 0.991 1.000 1.000 0.981 0.000 0.020 0.996 c 0.041 0.018 0.009 0.053 0.000 0.037 0.000 0.000 0.000 0.000 0.019 0.000 0.024 0.025 d 0.000 o.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 1.091 Got-2 a 0.007 0.000 0.009 0.021 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.013 0.014 b 0.972 1.000 0.991 0.979 1.000 1.000 1.000 1.000 1.000 1.000 0.981 1.000 0.019 0.017 c 0.021 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.019 0.000 0.018 0.018 Gpt-2 a 0.492 0.620 0.417 0.345 0.431 0.452 0.548 0.417 0.500 0.430 0.549 1.000 0.024 0.115 b 0.508 0.380 0.583 0.655 0.569 0.548 0.452 0.583 0.500 0.570 0.451 0.000 0.024 0.115 Pgm2 a 0.007 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.007 0.007 b 0.387 0.393 0.398 0.167 0.313 0.405 0.250 0.321 0.239 0.295 0.368 0.000 0.028 0.068 c 0.585 0.607 0.602 0.782 0.688 0.595 0.729 0.679 0.761 0.682 0.585 1.000 0.024 0.067 d 0.021 0.000 0.000 0.051 0.000 0.000 0.021 0.000 0.000 0.023 0.047 0.000 0.026 0.027 III.