Behavior Genetics, Vol. 9, No. 5, 1979
SHORT COMMUNICATION Inbreeding and Mating Patterns in Drosophila
pseudoobscura Jeffrey R. PowelF and Lois Morton 2 Received 26 May 1978--Final 20 Apr. 1979
Inbreeding, up to 12 generations o f single-pair matings, did not cause significant deviation from random mating among two sets o f strains in Drosophila pseudoobscura. This contrasts with reports that inbreeding D. melanogaster induces negative assortative mating among lines. KEY WORDS: random mating; Drosophila pseudoobscura; pheromones; Drosophila melano-
gaster.
The genetic basis of observable mating behavior is a very widely studied topic in behavior genetics, in part because it is an area in which the interests of behaviorists and population geneticists overlap. As with many areas of experimental population genetics, Drosophila has been utilized in numerous studies of the genetic control of mating behavior (see reviews by Spieth, 1952; Petit and Ehrman, 1969; Spiess, 1970). Averhoff and Richardson (1974), in a study of the mating behavior of inbred strains of Drosophila melanogaster, made a very striking observation. They found that with increasing generations of inbreeding, flies exhibited increasing negative assortative mating, i.e., matings within an inbred line became much less frequent than matings between lines when a choice was available. Negative assortative mating, which reached a maximum of 76% at eight generations of inbreeding, also paralleled the loss of inbred lines, suggesting that loss of lines after several generations of inThis research was supported by NSF Grant DEB-76-19971 and NIH Genetics Training Grant GM 07499. LDepartment of Biology, Yale University, New Haven, Connecticut 06520. 2 Department of Human Genetics, Yale University, New Haven, Connecticut 06520. 425 0001-8244/79/0900-0425503.00/0 9 1979 Plenum Publishing Corporation
426.
Powell and Morton
breeding may be due in part to nonmating rather than sterility or inviability associated with recessive lethals. Tests of "reluctant" males, either by subsequent pairings or by dissection of gonads, supported the conclusion that in most cases nonmating between sibs was not the result of male sterility. Averhoff and Richardson (1974) postulated that the loci controlling production and/or recognition of sex pheromones are polymorphic and, through inbreeding, different lines have "fixed" different alleles at one or more of these loci. They hypothesized that, because of receptor saturation, a male did not respond to pheromones which were abundant in his environment, i.e., those pheromones produced by the male himself and therefore by his very similar female sibs. It was only when confronted by a novel pheromone, in the form of a genetically different female, that the male became stimulated to mate. Averhoff and Richardson supported their hypothesis with data demonstrating that the frequency of male courtship of female sibs increased dramatically when the flies were exposed to pheromones produced by females of another line. Subsequently, the authors have shown that, in addition to this pheromonal signal, a second signal is required to induce female acceptance of male sibling courtship (Averhoff and Richardson, 1976). Yamazaki et aL (1978) reported preferential mating between several murine congenic lines differing only at 11-2 loci. However, both positive and negative assortative mating were observed. The authors favored a pheromortal hypothesis, but experiments proving an olfactory signal, such as those of Averhoff and Richardson (1974, 1976), were not performed. Such genetically determined inbreeding avoidance mechanisms, or "incest taboos," may have important population genetic consequences. Effective population size, and thus the extent of genetic variation, may be greater than commonly assumed. Segregational genetic load may also be reduced. Because of these implications we felt it was important to test the generality of the phenomenon especially in the genus Drosophila. We have studied two sets of strains of Drosophila pseudoobscura. Five lines were begun by single females collected from a natural population in the Central Highlands of Mexico at Amecameca. After they had been under laboratory culture about three or four generations, each line was single-pair (brother-sister) mated for 12 generations and tested after four, seven, and 12 generations. The other set of strains came from a highly heterogeneous population, designated "Origin," which had been maintained in a very large population cage for about 5 years. This population was begun from a dihybrid cross among strains from four geographically widely separated D. pseudoobscura populations (see Powell, 1978, for details). Eight lines from this population were single brother-sister mated for eight generations and tested after four and eight generations. For mating preference tests, we
Inbreeding and Mating Patterns in Drosophilapseudoobscura
427
collected virgin females and males and clipped the wings of some for identification. Twelve females and 12 males, 3 to 6 days after eclosion, of each of two strains were introduced without anesthetizing into an Elans-Wattiaux (1964) type mating chamber for direct observation of mating. Alternate strains were clipped in successive tests. As reported elsewhere (Ehrman, 1966; Powell, 1978), clipping the tips of D. pseudoobscura wings does not detectably affect mating. For the Amecameca strains we tested every possible pairwise combination of strains; for Origin strains we randomly chose 13 and nine combinations in the F4 and Fs generations, respectively. For each combination of two strains we observed a minimum of 50 matings; this usually meant four chambers, two with each strain clipped. Table I summarizes our results. None of the overall X~ tests indicates significant departure from random mating expectations. In addition, all of the individual combinations were tested for nonrandom mating. Only one combination (in Amecameca F4) yielded a x 2 significant at the 0.05 level. However, given that we computed x ~ for 43 combinations, we would expect one or two tests to be significant at the 0.05 level by chance alone. When the x2's are corrected for multiple tests by the method of Cooper (1968), none is significant. We conclude that inbreeding did not cause significant departure from random mating in the D. pseudoobscura populations we studied. These results differ from those of Averhoff and Richardson (1974, 1976); they observed preferences for mating between inbred lines as compared to within lines. They worked with the cosmopolitan, human commensal Drosophila melanogaster. Our study used D. pseudoobscura, which is confined to the western half of North America (and Bogot/t, Colombia) and breeds primarily in temperate oak-pine forests. Despite their narrower distribution, D. pseudoobscura populations are at least as genetically variable as D. melanogaster populations for both chromosome and allozyme Table I.
Results of Mating Tests Among Inbred Lines of Drosophila pseudoobscura ~
Number of lines
Possible number matings
Total number matings
Homogametic
Heterogametic
X12
Amecameca F4 . F7 F12
5 3 3
1104 432 288
633 287 182
293 159 94
340 128 88
3.49 3.35 0.20
Origin F4 Fs
8 6
1152 864
874 533
439 272
435 261
0.02 0.23
a None of the X~'s is significant at the 0.05 level. More detailed data for Origin F4 can be found in Powell (1978).
428
Powell and Morton
polymorphisms. Thus it seems unlikely that the phenomenon Averhoff and Richardson observed with D. rnelanogaster is a prerequisite for the maintenance of high levels of genetic variation. Several explanations for the discrepant results are possible. First, D. pseudoobscura may not be polymorphic for sex pheromone production. However, Leonard et al. (1974) have reported evidence to the contrary. Second, our original populations may not have been polymorphic at the relevant loci. But we chose to start our experiments with two genetically heterogeneous populations. Amecameca is very polymorphic for both chromosome and allozyme variation (unpublished observations). "Origin" is a hybrid population begun by a dihybrid cross among fresh collections from British Columbia, Washington State, and two locations in California (Powell, 1978). Thus, if genetic variation for sex pheromone production is common in D. pseudoobscura, it was probably present in our foundation populations. Third, it is possible that even though the initial populations were polymorphic at the loci controlling pheromone production, many of the inbred lines "fixed" the same allele. However, given that we began with two distinct foundation populations which yielded a total of 23 inbred lines, this seems unlikely. Finally, pheromonal control of mating behavior may not be important in D. pseudoobscura in the same manner as in D. melanogaster. Fixation of alternative alleles for pheromones in inbred lines may not affect mate choice. In a sense, the specific cause is less important than the finding of no significant departure from random mating. Whether the random mating we observed results from the failure of a mechanism for avoidance of close inbreeding or the absence of such a mechanism in the populations we studied, it demonstrates that incest taboos are not universal in Drosophila. REFERENCES Averhoff, W. W., and Richardson, R. H. (1974). Pheromonal control of mating patterns in Drosophila melanogaster. Behav. Genet. 4:207-225. Averhoff, W. W., and Richardson, R. H. (1976). Multiple pheromone system controlling mating in Drosophila melanogaster. Proc. Natl. Acad. Sci. 73:591-593. Cooper, D. W. (1968). The significance level in multiple tests made simultaneously. Heredity 23:614-617. Ehrman, L. (1966). Mating success and genotype frequency in Drosophila. Anim. Behav. 14:332-339. Elans, A. A., and Wattiaux, J. M. (1964). Direct observation of sexual isolation. Drosophila Inform. Serv. 39:118-119. Leonard, J. E., Ehrman, L., and Sehorsch, M. E. (1974). Bioassay of a Drosophila pheromone influencing sexual selection. Nature 250:261-262. Petit, C., and Ehrman, L. (1969). Sexual selection in Drosophila. Evol. Biol. 3:177-223. Powell, J. R. (1978). The founder-flush speciation theory: An experimental approach. Evolution 32:465-474.
Inbreeding and Mating Patterns in Drosophilapseudoobscura
429
Spiess, E. B. (1970). Mating propensity and its genetic basis in Drosophila. In Hecht, M. K., and Steere, W. C. (eds.), Essays in Evolution and Genetics in Honor of Th. Dobzhansky, Appleton-Century-Crofts, New York, pp. 315-380. Spieth, H. T. (1952). Mating behavior in the genus Drosophila (Diptera). Bull. Am. Mus. Nat. Hist. 99:395--474. Yamazaki, K., Yamaguchi, M., Andrews, P. W., Peake, B., and Boyse, E. A. (1978). Mating preferences of F2 segregants of crosses between MHC-congenic mouse strains. Immunogenetics 6:253-259. Edited by John M. Ringo
SHORT COMMUNICATION Inbreeding and Mating Patterns in Drosophila
pseudoobscura Jeffrey R. PowelF and Lois Morton 2 Received 26 May 1978--Final 20 Apr. 1979
Inbreeding, up to 12 generations o f single-pair matings, did not cause significant deviation from random mating among two sets o f strains in Drosophila pseudoobscura. This contrasts with reports that inbreeding D. melanogaster induces negative assortative mating among lines. KEY WORDS: random mating; Drosophila pseudoobscura; pheromones; Drosophila melano-
gaster.
The genetic basis of observable mating behavior is a very widely studied topic in behavior genetics, in part because it is an area in which the interests of behaviorists and population geneticists overlap. As with many areas of experimental population genetics, Drosophila has been utilized in numerous studies of the genetic control of mating behavior (see reviews by Spieth, 1952; Petit and Ehrman, 1969; Spiess, 1970). Averhoff and Richardson (1974), in a study of the mating behavior of inbred strains of Drosophila melanogaster, made a very striking observation. They found that with increasing generations of inbreeding, flies exhibited increasing negative assortative mating, i.e., matings within an inbred line became much less frequent than matings between lines when a choice was available. Negative assortative mating, which reached a maximum of 76% at eight generations of inbreeding, also paralleled the loss of inbred lines, suggesting that loss of lines after several generations of inThis research was supported by NSF Grant DEB-76-19971 and NIH Genetics Training Grant GM 07499. LDepartment of Biology, Yale University, New Haven, Connecticut 06520. 2 Department of Human Genetics, Yale University, New Haven, Connecticut 06520. 425 0001-8244/79/0900-0425503.00/0 9 1979 Plenum Publishing Corporation
426.
Powell and Morton
breeding may be due in part to nonmating rather than sterility or inviability associated with recessive lethals. Tests of "reluctant" males, either by subsequent pairings or by dissection of gonads, supported the conclusion that in most cases nonmating between sibs was not the result of male sterility. Averhoff and Richardson (1974) postulated that the loci controlling production and/or recognition of sex pheromones are polymorphic and, through inbreeding, different lines have "fixed" different alleles at one or more of these loci. They hypothesized that, because of receptor saturation, a male did not respond to pheromones which were abundant in his environment, i.e., those pheromones produced by the male himself and therefore by his very similar female sibs. It was only when confronted by a novel pheromone, in the form of a genetically different female, that the male became stimulated to mate. Averhoff and Richardson supported their hypothesis with data demonstrating that the frequency of male courtship of female sibs increased dramatically when the flies were exposed to pheromones produced by females of another line. Subsequently, the authors have shown that, in addition to this pheromonal signal, a second signal is required to induce female acceptance of male sibling courtship (Averhoff and Richardson, 1976). Yamazaki et aL (1978) reported preferential mating between several murine congenic lines differing only at 11-2 loci. However, both positive and negative assortative mating were observed. The authors favored a pheromortal hypothesis, but experiments proving an olfactory signal, such as those of Averhoff and Richardson (1974, 1976), were not performed. Such genetically determined inbreeding avoidance mechanisms, or "incest taboos," may have important population genetic consequences. Effective population size, and thus the extent of genetic variation, may be greater than commonly assumed. Segregational genetic load may also be reduced. Because of these implications we felt it was important to test the generality of the phenomenon especially in the genus Drosophila. We have studied two sets of strains of Drosophila pseudoobscura. Five lines were begun by single females collected from a natural population in the Central Highlands of Mexico at Amecameca. After they had been under laboratory culture about three or four generations, each line was single-pair (brother-sister) mated for 12 generations and tested after four, seven, and 12 generations. The other set of strains came from a highly heterogeneous population, designated "Origin," which had been maintained in a very large population cage for about 5 years. This population was begun from a dihybrid cross among strains from four geographically widely separated D. pseudoobscura populations (see Powell, 1978, for details). Eight lines from this population were single brother-sister mated for eight generations and tested after four and eight generations. For mating preference tests, we
Inbreeding and Mating Patterns in Drosophilapseudoobscura
427
collected virgin females and males and clipped the wings of some for identification. Twelve females and 12 males, 3 to 6 days after eclosion, of each of two strains were introduced without anesthetizing into an Elans-Wattiaux (1964) type mating chamber for direct observation of mating. Alternate strains were clipped in successive tests. As reported elsewhere (Ehrman, 1966; Powell, 1978), clipping the tips of D. pseudoobscura wings does not detectably affect mating. For the Amecameca strains we tested every possible pairwise combination of strains; for Origin strains we randomly chose 13 and nine combinations in the F4 and Fs generations, respectively. For each combination of two strains we observed a minimum of 50 matings; this usually meant four chambers, two with each strain clipped. Table I summarizes our results. None of the overall X~ tests indicates significant departure from random mating expectations. In addition, all of the individual combinations were tested for nonrandom mating. Only one combination (in Amecameca F4) yielded a x 2 significant at the 0.05 level. However, given that we computed x ~ for 43 combinations, we would expect one or two tests to be significant at the 0.05 level by chance alone. When the x2's are corrected for multiple tests by the method of Cooper (1968), none is significant. We conclude that inbreeding did not cause significant departure from random mating in the D. pseudoobscura populations we studied. These results differ from those of Averhoff and Richardson (1974, 1976); they observed preferences for mating between inbred lines as compared to within lines. They worked with the cosmopolitan, human commensal Drosophila melanogaster. Our study used D. pseudoobscura, which is confined to the western half of North America (and Bogot/t, Colombia) and breeds primarily in temperate oak-pine forests. Despite their narrower distribution, D. pseudoobscura populations are at least as genetically variable as D. melanogaster populations for both chromosome and allozyme Table I.
Results of Mating Tests Among Inbred Lines of Drosophila pseudoobscura ~
Number of lines
Possible number matings
Total number matings
Homogametic
Heterogametic
X12
Amecameca F4 . F7 F12
5 3 3
1104 432 288
633 287 182
293 159 94
340 128 88
3.49 3.35 0.20
Origin F4 Fs
8 6
1152 864
874 533
439 272
435 261
0.02 0.23
a None of the X~'s is significant at the 0.05 level. More detailed data for Origin F4 can be found in Powell (1978).
428
Powell and Morton
polymorphisms. Thus it seems unlikely that the phenomenon Averhoff and Richardson observed with D. rnelanogaster is a prerequisite for the maintenance of high levels of genetic variation. Several explanations for the discrepant results are possible. First, D. pseudoobscura may not be polymorphic for sex pheromone production. However, Leonard et al. (1974) have reported evidence to the contrary. Second, our original populations may not have been polymorphic at the relevant loci. But we chose to start our experiments with two genetically heterogeneous populations. Amecameca is very polymorphic for both chromosome and allozyme variation (unpublished observations). "Origin" is a hybrid population begun by a dihybrid cross among fresh collections from British Columbia, Washington State, and two locations in California (Powell, 1978). Thus, if genetic variation for sex pheromone production is common in D. pseudoobscura, it was probably present in our foundation populations. Third, it is possible that even though the initial populations were polymorphic at the loci controlling pheromone production, many of the inbred lines "fixed" the same allele. However, given that we began with two distinct foundation populations which yielded a total of 23 inbred lines, this seems unlikely. Finally, pheromonal control of mating behavior may not be important in D. pseudoobscura in the same manner as in D. melanogaster. Fixation of alternative alleles for pheromones in inbred lines may not affect mate choice. In a sense, the specific cause is less important than the finding of no significant departure from random mating. Whether the random mating we observed results from the failure of a mechanism for avoidance of close inbreeding or the absence of such a mechanism in the populations we studied, it demonstrates that incest taboos are not universal in Drosophila. REFERENCES Averhoff, W. W., and Richardson, R. H. (1974). Pheromonal control of mating patterns in Drosophila melanogaster. Behav. Genet. 4:207-225. Averhoff, W. W., and Richardson, R. H. (1976). Multiple pheromone system controlling mating in Drosophila melanogaster. Proc. Natl. Acad. Sci. 73:591-593. Cooper, D. W. (1968). The significance level in multiple tests made simultaneously. Heredity 23:614-617. Ehrman, L. (1966). Mating success and genotype frequency in Drosophila. Anim. Behav. 14:332-339. Elans, A. A., and Wattiaux, J. M. (1964). Direct observation of sexual isolation. Drosophila Inform. Serv. 39:118-119. Leonard, J. E., Ehrman, L., and Sehorsch, M. E. (1974). Bioassay of a Drosophila pheromone influencing sexual selection. Nature 250:261-262. Petit, C., and Ehrman, L. (1969). Sexual selection in Drosophila. Evol. Biol. 3:177-223. Powell, J. R. (1978). The founder-flush speciation theory: An experimental approach. Evolution 32:465-474.
Inbreeding and Mating Patterns in Drosophilapseudoobscura
429
Spiess, E. B. (1970). Mating propensity and its genetic basis in Drosophila. In Hecht, M. K., and Steere, W. C. (eds.), Essays in Evolution and Genetics in Honor of Th. Dobzhansky, Appleton-Century-Crofts, New York, pp. 315-380. Spieth, H. T. (1952). Mating behavior in the genus Drosophila (Diptera). Bull. Am. Mus. Nat. Hist. 99:395--474. Yamazaki, K., Yamaguchi, M., Andrews, P. W., Peake, B., and Boyse, E. A. (1978). Mating preferences of F2 segregants of crosses between MHC-congenic mouse strains. Immunogenetics 6:253-259. Edited by John M. Ringo
