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Proc. Nat. Acad. Sci. USAVol. 71, No. 3, pp. 999-1003, March 1974
Genetic Differentiation Within and Between Species of the Drosophilawillistoni Group*
(natural selection/evolution/adaptation/isozymes/Caribbean islands)
FRANCISCO J. AYALA AND MARTIN L. TRACEY
Department of Genetics, University of California, Davis, Calif. 95616
Communicated by Theodosius Dobzhansky, October 2, 1973
ABSTRACT We describe allelic variation at 28 loci insix Caribbean populations of four sympatric species ofDrosophila. Within any one species the allelic frequenciesare very similar from population to population, althoughthere is evidence of local as well as regional genetic dif-ferentiation. The genetic distance is greater betweenpopulations from different islands than between popula-tions of the same island. When the allelic frequencies arecompared between different species, a remarkable patternappears. In any pair of species nearly half of the loci haveessentially identical allelic frequencies, while nearly theother half of the loci have different alleles and in differentfrequencies. The loci with nearly identical allelic fre-quencies are different when different pairs of species arecompared. The patterns of allelic variation within andbetween species are inconsistent with the hypothesisthat the variation is adaptively neutral. Migration ormutation cannot explain the patterns of genetic variation,either. Balancing natural selection is the main processmaintaining protein polymorphisms in natural popula-tions.
Four species of the Drosophila willistoni group are sympatricin the Greater Antilles. Three species are siblings, D. willistoniSturtevant, D. tropicalis Burla and de Cunha, and D. equi-noxialis caribbensis Ayala. The fourth, D. nebulosa Sturtevant,is a close relative of the other three, but can be easily distin-guished from them by external morphology. We have studiedallelic variation in 28 genes coding for enzymes in samples ofthese species collected in two localities in Hispaniola, three inPuerto Rico, and one in St. Kitts, one of the Lesser Antilles.We report our results concerning the amount of genetic varia-tion within local populations, between local populations in thesame island, between populations of different islands, andbetween different species. The pattern of genetic variationwithin and between species is inconsistent with the notionthat the variation is adaptively neutral.
MATERIALS AND METHODS
Flies of the D. willistoni group were collected over bananabaits and naturally occurring decaying fruits in late Februaryand early March, 1972, in the following localities. DominicanRepublic: Santiago, about 10 km NE of that city, on the foot-hills of the Cordillera Septentional; Santo Domingo, about25 km NW of that city, on the southeastern slopes of theCordillera Central; Puerto Rico: Mayagfiez, in the westernend of the island on the grounds of the U.S.D.A. ExperimentalStation; Barranquitas, 2-5 km from that town, in a smallforest (less than 0.01 kM2) within the grounds of the home ofDr. Roberto Aponte, M.D., on the Cordillera Central;Yunque, in the rain forest known by that name, near the
place called La Mina, Sierra de Luquillo, near the easterntip of the island. St. Kitts: on a small gallery forest above thetown of Canyon, on the southeastern slopes of Mt. Misery.D. willistoni was collected in all six localities; D. tropicalis andD. e. caribbensis in all but St. Kitts; D. nebulosa in all exceptYunque.Our techniques of starch-gel electrophoresis and enzyme
assay are described elsewhere (1). The 28 gene loci and theenzymes they code for are as follows: esterases (EC 3.1.1.2),six loci (Est-2, Est-S, Est-4, Est-5, Est-6, Est-7); TPN+-de-pendent malate dehydrogenase (EC 1.1.1.40), two loci (Me-i,Me-2); adenylate kinase (EC 2.7.4.3), two loci (Adk-1, Adk-2);hexokinase (EC 2.7.1.1), three loci (Hk-1, Hk-2, Hk-3); andone locus for each of the following: leucine aminopeptidase(EC 3.4.1.1) (Lap-5), alkaline phosphatase (EC 3.1.3.1)(Aph-1), acid phosphatase (EC 3.1.3.2) (Acph-1), aldolase(EC 4.1.2.13) (Ald), alcohol dehydrogenase (EC 1.1.1.1)(Adh), malate dehydrogenase (EC 1.1.1.37) (Mdh-2), a-glyc-erophosphate dehydrogenase (EC 1.1.9.5) (aGpdh), isoci-trate dehydrogenase (EC 1.1.1.41) (Idh), glyceraldehyde-3-phosphate dehydrogenase (EC 1.1.1.8) (G3pdh), octanol de-hydrogenase (EC 1.1.1.1) (Odh-1), xanthine dehydrogenase(EC 1.2.3.2) (Xdh), aldehyde oxidase (EC 1.2.3.1) (Ao-1),tetrazolium oxidase (To), triose phosphate isomerase (EC1.2.1.9) (Tpi-2), and phosphoglucomutase (EC 2.7.5.1)(Pgm-1).Wild males were used directly for electrophoresis. Individual
females were placed in separate culture bottles; one F1 femalewas run from each culture for a given enzyme.Numbers are used to refer to the alleles. At each locus, one
allele is taken as standard, and named 100. The other allelesare designated by adding or subtracting from 100 the dif-ference in anodal migration, expressed in millimeters, be-tween the enzyme coded by each allele and the standard.
RESULTS AND DISCUSSION
The allelic frequencies at each of 27 loci are shown in Table 1for the six populations of D. willistoni. The sample size is thenumber of wild genomes studied. The proportion of heterozy-gotes given is the value expected according to the Hardy-Weinberg principle; the observed and expected frequenciesgenerally agree quite well.As it has been reported earlier in natural populations of
D. willistoni and other species of the group (1-5), the patternsof the allelic frequencies are very similar in all localities.Generally, at any one locus the same allele is the most fre-quent in every population, and the same alleles appear atintermediate and low frequencies. Nevertheless, at some locithere are significant differences between localities in the allelic
* This is paper no. 9 in a series: "Enzyme variability in theDrosophila willistoni group." Paper no. 5 is ref. 3.
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TABLE 1. Allelic variation at 27 loci in natural populations TABLE 1. Allelic variation at 27 loci in natural populationsof Drosophila willistoni of Drosophila willistoni
Santo Barran- Santo Barran-Gene Alleles Santiago Domingo Mayag,3ez quitas Yunque St. Kitts Gene Alleles Santiago Domingo Mayasuez quOtas Yunque St. Kitts
Lap-5 Sample size 320 33694 .000 .00096 .000 .00698 .044 .057
100 .597 .494103 .338 .429105 .022 .015
Heterozygotes .527 .569
Est-2 Sample size 320 33298 .000 .006100 .181 .175102 .813 .807104 .006 .009106 .000 .003
Heterozygotes .307 .318
Est-3 Sample size 320 33698 .025 .012100 .963 .985102 .013 .003
Heterozygotes .073 .029
Est-4 Sample size 322 33698 .003 .000102 .991 .997104 .006 .003
Heterozygotes .018 .006
Est-5 Sample size 318 33690 .000 .00095 .000 .000
100 1.000 1. 000105 .000 .000
Heterozygotes .000 .000
Est-7 Sample size 158 16892 .000 .00694 .000 .00095 .006 .00096 .025 .01898 .120 .119
100 .525 .530102 .291 .274105 .032 .054107 .000 .000120 .000 .000
Heterozygotes .623 .627
Aph-l Sample size 80 17098 .000 .000
100 .950 .953102 .025 .012104 .025 .035
Heterozygotes .096 .091
Acph-l Sample size 322 32288 .000 .00094 .006 .01997 .003 .000100 .988 .978104 .003 .003106 .000 .000
Heterozygotes .025 .043
Ald Sample size 198 15696 .000 .00098 .000 .013100 .677 .647102 .303 .333104 .000 .006105 .020 .000
Heterozygotes .450 .470
Adh Sample size 316 33690 .000 .003
100 .997 .99.4106 .003 .003
Heterozygotes .006 .012
Mdh-2 Sample size 198 16686 .000 .00688 .000 .00094 .298 .404100 .702 .584104 .000 .000106 .000 .006112 .000 .000
Heterozygotes .418 .496
aPpdh Sample size 322 33288 .000 .00094 .003 .003100 .997 .997106 .000 .000
Reteroxygotes .006 .006
Idh Sample size 198 16692 .005 .00096 .010 .000
100 .985 .994104 .000 .006108 .000 .000
Heterozygotes .030 .012
G3pdh Sample size 198 16696 .010 .00098 .005 .018
100 .944 .958102 .005 .024105 .035 .000
Heterozygotes .107 .082
458 624 498 364.000 .000 .004 .000.004 .005 .000 .003.039 .032 .014 .008.664 .736 .801 .810.282 .223 .177 .170.011 .005 .004 .008.478 .408 .327 .314
454 636 502 372.004 .003 .024 .032.110 .129 .129 .132.881 .855 .837 .833.004 .013 .010 .003.000 .000 .000 .000.212 .252 .283 .287
372 628 424 328.013 .013 .026 .015.984 .976 .972 .985.003 .011 .002 .000.032 .047 .055 .030
456 634 466 372.007 .000 .002 .000.985 .992 .991 1.000.009 .008 .006 .000.030 .016 .017 .000
458 650 500 372.000 .002 .000 .000.002 .002 .002 .000.998 .994 .992 1.000.000 .003 .006 .000.004 .012 .016 .000
229 313 251 186.000 .000 .004 .000.000 .000 .004 .005.009 .000 .008 .000.022 .013 .012 .016.118 .096 .139 .145.576 .562 .530 .575.236 .256 .243 .220.035 .061 .048 .032.004 .013 .008 .005.000 .000 .004 .000.596 .605 .638 .598
186 144 168 20.032 .014 .012 .000.871 .917 .869 1.000.032 .069 .107 .000.065 .000 .012 .000.235 .155 .232 .000
456 684 618 372.002 .003 .000 .000.011 .015 .011 .000.000 .003 .000 .000.987 .978 .982 .989.000 .000 .000 .003.000 .001 .006 .008.026 .043 .035 .021
136 374 426 218.000 .003 .000 .000.000 .011 .005 .000.713 .834 .660 .651.279 .150 .336 .349.007 .003 .000 .000.000 .000 .000 .000.4.3 .282 .452 .454
422 662 506 364.000 .002 .002 .000.993 .995 .998 1.000.007 .003 .000 .000.014 .009 .004 .000
226 470 192 198.000 .002 .000 .000.000 .004 .000 .000.217 .200 .214 .000.774 .794 .781 1.000.000 .000 .005 .000.004 .000 .000 .000.004 .000 .000 .000.353 .330 .344 .000
458 682 510 370.000 .000 .000 .003.002 .000 .000 .000.998 .997 1.000 .992.000 .003 .000 .005.004 .006 .000 .016
212 482 436 220.000 .000 .000 .000.005 .008 .002 .000.995 .992 .991 .995.000 .000 .005 .005.000 .000 .002 .000.009 .016 .018 .009
228 342 436 176.026 .029 .030 .0 34.000 .000 .000 .000.934 .956 .947 .938.000 .000 .000 .000.039 .015 .023 .028.125 .085 .101 .119
Odh-l Sample size 188 16686 .000 .00094 .032 .03096 .000 .000
100 .952 .934104 .011 .024108 .005 .012
Heterozygotes .092 .127
Me-i Sample size 188 16492 .000 .00096 .005 .012
100 .979 .976104 .016 .012
Heterozygotes .042 .047
Me-2 Sample size 198 14692 .010 .00096 .035 .021100 .753 .637104 .202 .342108 .000 .000
Heterozygotes .392 .477
ILh Sample size 320 24696 .000 .00497 .006 .00098 .125 .11499 .088 .089
100 .484 .585101 .269 .203102 .025 .004103 .003 .000104 .000 .000
Heterozygotes .669 .595
Ao-i Sample size 70 6295 .000 .03296 .000 .01698 .100 .081
100 .886 .774102 .014 .097103 .000 .000105 .000 .000
lSeterozygotes .205 .383
To Sample size 260 19686 .000 .01094 .000 .000
100 1.000 .980108 .000 .010
Heterozygotes .000 .040
Tpi-2 Sample size 198 16692 .000 .00094 .000 .000
100 1.000 1.000106 .000 .000
Heterozygotes .000 .000
Pgm-1 Sample size 198 16480 .000 .00096 .025 .024100 .960 .976104 .015 .000108 .000 .000
Heterozygotes .078 .048
Adk-l Sample size 198 11894 .015 .034100 .212 .314106 .636 .593112 .126 .051118 .010 .008
Heterozygotes .534 .546
Adk-2 Sample size 198 16496 .000 .006100 .985 .988104 .010 .006108 .005 .000
Heterozygotes .030 .024
Hk-i Sample size 198 16296 .025 .019100 .864 .914104 .111 .068108 .000 .000
Heterozygotes .241 .160
Hk-2 Sample size 198 16092 .000 .00096 .000 .000100 .934 .538104 .020 .012108 .045 .450
Heterozygotes .125 .508
Hk-3 Sample size 198 16492 .005 .00696 .005 .037
100 .985 .927104 .005 .030108 .000 .000
Heterozygotes .030 .139
110 372.000 .000.000 .000.009 .000.973 .978.009 .016.009 .005.053 .042
228 480.009 .000.018 .006.969 .985.004 .008.060 .029
182 346.000 .003.016 .020.868 .801.110 .173.005 .003.234 .329
342 592.006 .000.003 .007.079 .140.094 .074.404 .515.365 .253.044 .010.006 .000.000 .000.687 .645
- 54- .000- .000- .093- .833- .000- .019- .056- .294
294 508.000 .006.000 .000
1.000 .994.000 .000.000 .012
228 480.000 ..002.000 .002
1.000 .996.000 .000.000 .008
228 482.000 .002.004 .002.961 .981.035 .015.000 .000.076 .037
202 316.000 .009.277 .266.609 .639.114 .079.000 .006.539 .514
228 472.009 .009.965 .983.018 .004.009 .004.068 .034
228 480.004 .008.930 .927.061 .060.004 .004.132 .137
228 472.000 .000.009 .004.956 .936.026 .019.009 .040.085 .121
228 478.000 .002.004 .004.982 .979.013 .006.000 .008.035 .041
334 112.003 .000.006 .000.003 .000.961 .991.023 .009.003 .000.076 .018
434 218.000 .000.005 .005.991 .991.005 .005.018 .018
428 210.000 .000.042 .014.743 .762.210 .219.005 .005.402 .371
502 318.002 .000.004 .022.092 .126.034 .050.446 .374.406 .418.014 .009.000 .000.002 .000.626 .666
457 292.009 .003.002 .000.989 .997.000 .000.022 .007
428 220.000 .000.000 .000.998 .995.002 .005.005 .009
434 218.000 .000.014 .000.965 .963.021 .032.000 .005.067 .071
404 220.000 .000.309 .177.574 .736.114 .086.002 .000.562 .419
422 218.005 .000.943 .954.043 .046.009 .000.109 .088
420 220.000 .009.979 .964.021 .023.000 .005.042 .071
430 220.002 .000.014 .009.895 .891.063 .082.026 .018.194 .199
430 220.002 .000.009 .005.967 .968.021 .027.000 .000.064 .062
frequencies, for instance, at the loci Lap-5, Ald, Mdh-2, Xdh,and Hk-2 (Table 1).
Comparison of the data in Table 1 with published results(1, 2, 5) shows that differentiation occurs also between regions
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Proc. Nat. Acad. Sci. USA 71 (1974)
TABLE 2. Allelic variation at 28 loci in natural populationsof four species of the Drosophila willistoni group
Genetic Differentiation 1001
TABLE 2. Allelic variation at 28 loci in natural populationsof four species of the Drosophila willistoni group (Cont'd.)
Gene Alleles D. willistoni D. tropicalis D. e. caribbensis D. nebulosa Gene Alleles D. willistoni D. tropicalis D. e. caribbensis D. nebulosa
Lap-5 Sample size98
100103105107109
Heterozygotes
Est-2 Sample size9698
100102
Heterozygotes
Est-3 Sample size9798
100102
Heterozygotes
Est-4 Sample size98100102
Heterozygotes
Est-5 Sample size100105
Heterozygotes
Est-6 Sample size104106108
Heterozygotep
Est-
Acph
Aid
Adh
Mdh-2
aGpdh
Idh
G3ydh
Odh-1
Me-I
2600.031.698.258.010.000.000
.437+.043
2616.000.011.138.848
. 277+.016
2408.000.017.977.006
.044+.007
2586.002.000.992
.015+.004
2634.997.002
.005+.003
Not assayed
526.002.008.120.848.021.000
.254+.017
544.976.002.013.000
.058+.013
506.000.000.972.000
.058_.007
544.004.987.009
.021+.006
No activity
Not assayed
-7 Sample size 1305 27296 .017 .01198 .121 .121
100 .552 .449102 .251 .324105 .045 .074107 .006 .011
Heterozygotes .615+.007 .669+.011
-1 Sample size 768 180100 .909 .056102 .049 .900104 .029 .044
Heterozygotes .135+.037 .192+.071
-1 Sample size 2774 54288 .001 .00994 .011 .980
100 .983 .009106 .003 .000108 .000 .000
Heterozygotes .032+.004 .043+.007
Sample size 1508 350100 .708 .686102 .282 .291
Heterozygotes .420+.029 .404+.055
Sample size 2606 438100 .996 .998110 .000 .000
Heterozygotes .008+.002 .003+.003
Sample size 1450 35296 .001 .99494 .214 .00398 .000 .000
100 .781 .003106 .001 .000
Heterozygotes .324+.069 .007+.007
Sample size 2674 546100 .997 .993
Heterozygotes .006+.002 .014+.009
Sample size 1714 338100 .992 .997104 .002 .003
Heterozygotes .016+.003 .006+.006
Sample size 1546 30096 .024 .017
100 .947 .940105 .023 .023
Heterozygotes .103+.007 .140+.043
Sample size 1282 258100 .965 .953104 .017 .027
Heterozygotes .068+.016 .071+.043
Sample size 1712 24094 .000 .979
100 .984 .008104 .008 .000
Heterozygotes .036+.007 .049+.031
1564.000.000.000.000.757.230
.373+.007
1326.006.045.049.873
.326+.060
736.000.014.976.010
.107+.058
1430.986.007.001
.072+.026
1550.366.620
.493+.007
288.698.090.134
.496+.037
No activity
650.028.945.025
.105+.026
1610.563.017.145.226.012
.507+.074
694.729.255
.406+.043
1462.997.000
.032+.027
934.000.000.000.012.986
.023+.014
1708.999
.006+. 003
970.997.001
.020+.014
754.786.182.000
.108+.013
786.989.008
.034+.015
942.000.000.997
.013+.009
214.028.000.762.051.014.019
.420+.021
308.068.860.010.000
.209+.058
140.964.000.007.007
.086+.002
316.987.003.000
.017+.009
No activity
314.809.010.140
.300+.025
161.000.000.006.006.006.932
.134_.052
16.000.125.875.219
312.000.000.000.199.782
.402+.056
176.886.102
.221_.065
308.003.987
.031+.009
176.000.000.994.000.006.000
314.987
.014+.014
172.919.058
.183+.043
Not assayed
1521.000.000.000
176.000.028.966
.068+.020
Me-2 Sample size96
100104106108
Heterozygotes
xdh Sample size939596979899
100101102
Heterozygotes
Ao-l Sample size98
100102105
Heterozygotes
To Sample size100120
Heterozygotes
Tpi-2 Sample size100105106
Heterozygotes
Pgm-1 Sample size9698
100104
Heterozygotes
Adk-l Sample size100106112124
Heterozygotes
Adk-2 Sample size100104
Heterozygotes
Hk-l Sample size96
100104
Heterozygotes
k-2 Sample size100104108
Heterozygotes
Hk-3 Sample size100104
Heterozygotes
Sowm rare alleles are omitted from the table. Populations with sample sizesmaller than 20 have not been used to estimate the average frequency ofheterozygotes in the species.
at some loci of D. willistoni. At the Lap-5 locus, allele 103 isthe most frequent, followed in frequency by allele 100, in allcontinental populations of D. willistoni studied (1, 2). In thepresent samples from the Caribbean islands, as well as in sixother islands from the Lesser Antilles (2), allele 100 is themost frequent, and allele 103 the second most common.Another example of notable regional differentiation exists atthe ilidh-2 locus. Allele 94 is very rare in every populationpreviously studied, including those from the Lesser Antilles;its frequency in 7584 wild genomes previously sampled is0.011, while the frequency of allele 100 is 0.981 (1, 6). Thefrequencies of M71dh-294 and Mlfdh-2100 in St. Kitts agree withthe frequencies found in previous studies. In Puerto Rico andthe Dominican Republic, however, Mdh-294 has a frequencyabout 20 or more times greater (between 0.200 in Barran-quitas and 0.404 in Santo Domingo) than it has in all otherpopulations studied.
1510.027.765.203.000.003
.368+.033
2320.000.000.002.007.114.069.468.322.017
.648+.014
186.091.833.038.016
.294+.051
2007.994.000
.014+.006
1720.998.000.001
.004+.002
1724.010.000.969.020
.063+.007
1458.263.628.099.000
.519_.021
1702.968.022
.059_.014
1708.009.936.053
.131+.029
1708.885.039.069
.205+.063
1718.971.016
.062_.016
184.005.179.087.000.723
.322+.096
512.387.523.020.006.000.000.000.000.000
.553_.029
106.038.849.057.028
.234+.099
4201.000.000.000
356.997.000.000
.004+.004
352.009.000.099.807
.322_.044
344.340.573.081.000
.543+.018
354.986.006
.029+.012
352.009.969.023
.057+.007
352.835.043.114
.243+.068
352.980.014
.049+.018
900.020.876.099.000.003
.223+.023
1626.000.000.000.000:007.030.476.438.049
.574+.024
456.009.195.020.706
.195+.028
1313.998.000
.003+.001
968.001.000.996
.025+.012
964.002.000.058.933
.203+.064
770.140.742.110.000
.418+.036
968.001.979
.078+.046
966.982.008.000
.046+.011
968.937.014.043
.164_.057
960.978.011
.047_.010
166.000.000.012.940.024
.121+.045
294.000.058.711.167.034.000.000.000.000
.484+.063
Not assayed
145.000.986
.019+.019
176.000.989.000
.015+.015
174.810.155.006.000
.327+.065
174.000.138.644.161
.540+.011
174.006.971
.050_.020
174.000.138.862
.237+.041
176.898.006.091
.163+.075
1761.000.000.000
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1002 Genetics: Ayala and Tracey
50
40
0-i
zLLU
a.
30 F
201-
0
50
40
0
-J
z
LU
w
0LJ
301
20
10
I ,,i , r0. 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
GENETIC SIMILARITY
I I
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
GENETIC SIMILARITYFIG. 1 (top). Histogram showing the distribution of loci with
respect to genetic similarity, I, when pairs of sibling species are
compared.FIG. 2 (bottom). Histogram showing the distribution of loci
with respect to genetic similarity, I, when pairs of nonsiblingspecies are compared.
The patterns of genetic variation in the other three species,D. tropicalis, D. e. caribbensis, and D. nebulosa, are similar tothose found in D. willistoni. At each locus, the same allelesare found with roughly the same frequencies in all local popu-lations of a given species, although local and regional dif-ferentiation occur at some loci. As a measure of genetic dif-ferentiation, we have used the statistics I ("genetic similar-ity") and D ("genetic distance") as defined by Nei (7). Themean values of I between local populations are: D. willistoni,0.952 i 0.006; D. tropicalis, 0.949 +t 0.010; D. e. caribbensis,0.954 0.011; and D. nebulosa, 0.991 +4 0.002. In every spe-
cies, populations from the same island are genetically more
similar than populations from different islands, but differencesbetween the mean Is are statistically significant only in D.willistoni, I within islands = 0.980 + 0.007, I between is-lands = 0.942 + 0.005. A summary of genetic variation inthe four species is given in Table 3.
Table 2 gives the allelic frequencies in the four species for28 loci when the data for all local populations are pooledwithin each species. Some rare alleles have been omitted. Aremarkable pattern appears. Generally, when two species are
compared at any one locus, the configuration of allelic fre-quencies is either essentially identical (I > 0.95) or very dif-ferent (I < 0.05). The species that are similar vary fromlocus to locus. At six loci (four weakly polymorphic, aiGpdh,Idh, Odh-1, and Hk-3, and two moderately polymorphic,Ald and Hk-2), the four species are genetically very similar.
TABLE 3. Summary of genetic variation in naturalpopulations of four species of Drosophila
D. willistoni D. tropicalis D. e. caribbeasits D. nebulosa
Number of 27 26 27 25loci studied
Genes sampled 18.50 i 121 370 + 24 1048 + 73 203 i 15per locus
Polymorphic lociper population:
(1) 0.680 0.079 0.678 i 0.072 0.674 0.075 0.687 0.081(2) 0.4454±0.087 0.438 0.092 0.470 0.086 0.510 0.022
Heterozygous loci 0.182 0.039 0.168 0.038 0.189 0.036 0.170 0.033per individual
A locus is considered polymorphic (1) when the frequency ofthe second most common allele > 0.01; (2) when the frequency ofthe most common allele <0.95. The proportion of polymorphicloci per population and of heterozygous loci per individual havebeen calculated by estimating the appropriate parameter for eachlocus, and averaging these values over all loci.
At four loci (Lap-5, Est-5, Acph-1, and Mdh-2, all moderatelyor highly polymorphic in one or more species), no two specieshave similar patterns of allelic frequencies. At the other 18loci, one pair, two pairs, or a triplet of species are very similar,while the others are quite different.The pattern of variation of allelic frequencies is most sig-
nificant when any two species are compared through all locistudied in both species. Consider the pair, D. tropicalis andD. e. caribbensis. At 11 loci (Est-3, Aph-1, Ald, Adh, aGpdh,Idh, Odh-1, To, Pgm-1, Hk-2, and Hk-3) these two specieshave a genetic similarity, I, greater than 0.95, and at one
more locus (Adk-1) I > 0.90. On the other hand, at 10 loci(Lap-5, Est-2, Est-4, Acph-1, M1-dh-2, Mle-i, Xdh, Tpi-2, Adk-2,and Hk-1) these two species have very different configurationsof allelic frequencies, I < 0.05. Only three loci (G3pdh, JMle-2,and Ao-1) fall between these two extremes. A similar pictureemerges when either D. tropicalis or D. e. caribbensis is com-
pared with D. willistoni. When any of the three sibling speciesis compared with D. nebulosa most loci also have extremevalues of I, although the lprol)ortion of loci at which I < 0.05is greater and that at which I < 0.95 is smaller than whensibling species are compared. The distribution of loci fallingwithin each interval of I is shown in Fig. 1 for comparisonsbetween siblings, and in Fig. 2 for comparisons between non-
sibling species. The actual I values for each comparison ofpairs are given in Table 4. The mean I for comparisons be-tween sibling species is 0.546 + 0.040 (D = 0.610 0.071);and for comparisons between nonsiblings it is 0.334 0.048(D = 1.115 0.135).What processes are responsible for the observed slatterns
of allelic frequencies? It has been suggested that most or allgenetically controlled lrotein lpolymorlphisms are adaptivelyneutral (8, 9). This theory predicts that genetically isolatedpol)ulations should have at a given locus different alleles and.in different frequencies. If the protein variation is adaptivelyneutral, the occurrence of the same alleles in similar frequen-cies in two different l)ol)ulations could be explained by
TABLE 4. Genetic similarity, 1,in comparisons between pairs of species
D. tropicalis D. e. caribbensis D. nebulosa
D. willistoni 0.623 0.527 0.287D. tropicalis 0.489 0.286D. e. caribbensis 0.429
Proc. Nat. Acad. Sci. USA 71 (1974)
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Proc. Nat. Acad. Sci. USA 71 (1974)
assuming that gene flow exists between the pol)ulations (9)or, alternatively, by assuming that the populations becamegenetically isolated only recently and there has been insuffi-cient time for their divergence by random drift processes.These two alternatives can be discarded in light of the presentevidence. The lack of gene flow between these species is wellauthenticated (10). If there were sufficient gene flow betweentwo species to keep the allelic frequencies similar at a givenlocus, the allelic frequencies should be equally similar at allother neutral loci as well. Yet, in each pair of species the con-figuration of allelic frequencies is very similar at nearly halfof the loci, and very different at nearly the other half.
Similarities between pairs of species at many loci cannot beexplained, either, as due to lack of sufficient number of gen-erations for divergence of allelic frequencies. Allelic frequencieshave become in fact completely different at about half theloci, on theaverage, between any pair of species.A conceivable suggestion is that some loci have become
different in a relatively short time due to natural selection,while adaptively neutral loci have remained quite similarbecause not enough time has passed since the species becamereproductively isolated. However, most loci that have similarallelic frequencies in one pair of species have very differentallelic frequencies in some other pair. Only 6 out of 28 locistudied have similar allelic frequencies in all four species, andfour of them are essentially monomorphic. This unlikely sug-gestion would, in any case, require that strong natural selec-tion operate at 22, or 79%, of the loci surveyed.The opposite alternative is also conceivable; namely, that
similar configurations of allelic frequencies are maintained bynatural selection at some loci, while other loci have becomedifferentiated as the result of random drift of neutral allelesover many generations. All four species differ substantiallyfrom each other in the allelic frequencies at only four loci:Lap-5, Est-5, Acph-1, and Mfdh-2; at all other loci, there isat least one pair of species with very similar allelic frequencies.But even those four loci would have to be excluded as beingadaptively neutral when other lopulations of these and closelyrelated species are taken into consideration. The allelic fre-quencies are essentially identical: (1) at the Lap-5 locus incontinental populations of D. willistoni and of D. tropicalis(5); (2) at the Est-5 locus in continental populations of D.willistoni and of D. equinoxialis (1, 3); (3) at the Acph-1locus in populations of D. nebulosa, D. paulistorum, and D.insularis (the latter two are siblings of D. willistoni); and (4)at the Mdh-2 in continental populations of D. equinoxialisand D. paulistorum.Another conceivable explanation is that alleles are adap-
tively equivalent, but their frequencies are similar in dif-ferent species owing to mutation pressure. Mutations tocommon alleles would occur more frequently than mutationsto rare alleles. Allozymes with a given electrophoretic mo-bility do not necessarily have identical aminoacid sequences.That is, electrophoretically detectable alleles need not besingle alleles, but rather they may be classes of alleles codingfor different polypeptides with identical electrophoreticmobilities. Therefore, the frequency of an electrophoretic"allele" might represent the number of possible differentalleles within that mobility class. If the number of alleleswithin each class is large, genetically isolated populationscould nevertheless have similar frequency distributions of themobilitv classes, even though they might be adaptivelyequivalent. The distribution of allelic frequencies in the four
species rules out this possibility, since all four species havesimilar allelic frequencies at only 6 of the 28 loci.
Figs. 1 and 2 show the distribution of loci according to theirgenetic similarity (I) values in comparisons between l)airs ofspecies. Both figures show U-shaped distributions, with mostloci (83% in either figure) in the 0-0.05 or 0.95-1.00 classes.If allelic variation at these loci were adaptively neutral theirdistribution should be approximately normal around themean I value (11). Consider two poI)ulations that, at a giventime, had identical allelic frequencies at all loci but have beenevolving independently for a given length of time. Assumealso that at each locus there are several allelic states adap-tively equivalent. The probability of a given allele to increaseor decrease in frequency by a given amount each generationwould be the same for all alleles at all loci since it would bea function of the effective size of the population. If there weremany alleles at each locus the allelic frequencies in the twopopulations would continuously diverge at every locus. Thatis, the value of I would gradually decrease at a rate that hasthe same probability at each locus. After a given length oftime, the values of I for all loci would have a normal distribu-tion around their mean value. Clearly, the distributions inFigs. 1 and 2 are not normal at all. A conceivable escape fromthis argument is to suggest that at some loci natural selectionkeeps allelic frequencies similar, while the rest of the loci areevolving at random and the mean I for these loci is alproach-ing zero. However, this explanation is unsatisfactory since dif-ferent sets of loci fall in the 0.95-1 class when different pairsof species are compared. Thus the distribution of I given byFigs. 1 and 2 is incompatible with the hypothesis that allelicvariation at the loci studied is adaptively neutral. It shouldbe lointed out that if the allelic variation is adaptively neu-tral, the distribution of I should also be normal even whenmany alleles exist within each mobility class.The Ieattern of allelic variation in different local populations
of a given sl)ecies of the D. willistoni group has been shownelsewhere (1-3, 5) to be incoml)atible with the hypothesisthat l)rotein l)olymorl)hisms are adaptively neutral. Thisconclusion is further confirmed by the l)attern of allelic varia-tion in different species of the group. Balancing natural selec-tions (heterotic or frequency dependent) are the most likelyprocesses to account for the patterns of genetic variationobserved in natural l)o)ulations. Needless to say, allelicfrequencies determined by natural selection are also affectedby random drift, particularly in small populations.We thank -Miss Lorraine G. Barr for excellent technical
assistance. Prof. Th. Dobzhansky read the manuscript andprovided valuable advice and criticism.1. Ayala, F. J., Powell, J. R., Tracey, AI. L., -Mourdo, C. A. &
PWrez-Salas, S. (1972) Genetics 70, 113-139.2. Ayala, F. J., Powell, J. R. & Dobzhansky, Th. (1971) Proc.
Nat. Acad. Sci. USA 68, 2480-2483.3. Ayala, F. J., Powell, J. R. & Tracey, Al. L. (1972) Genet.
Res. 20, 19-42.4. Richmond, 1?. C. (1972) Genietics 70, 87-112.5. Ayala, F. J. (1972) Proc. Sixth Berkeley Symp. Math.
Stat. Prob. V, 211-236.6. Ayala, F. J. & Anderson, W. W. (1973) Nature New Biol.
241, 274-276.7. Nei, AI. (1972) Amer. Natur. 106, 283-292.8. King, J. L. & Jukes, T. H. (1969) Science 164, 788-798.9. Kimura, Ml. & Ohta, T. (1971) Nature 229, 467-469.
10. Spassky, B., Richmond, R. C., Perez-Salas, S., Pavlovsky,O., M\ourdo, C. A., Hunter, A. S., Hoeningsberg, H.,Dobzhansky, T. & Ayala, F. J. (1971) Evolution 25, 129-143.
11. Ayala, F. J. & Gilpin, M. E., manuscript in preparation.
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