And Tobari1969a, 196913; Kojimaand YARBROUGH1967), and Fecundity (Andersonand WATANABE1974) Components of Fitness

Home , Heterozygote advantage, List of polymorphisms

MODES OF SELECTION MAINTAINING AN INVERSION POLYMORPHISM IN DROSOPHILA PAULZSTORUM

MARK H. GROMKO AND ROLIJN C. RICHMOND Department of Zoology, Indiana University, Bloomington, Indiana 47401 Manuscript received July 6,1976 Revised copy received October, 1977

ABSTRACT

The possibility that fitness relationships associated with an inversion polymorphism in D. paulisforum were frequency dependent was investigated. Using allozymes of tetrazolium oxidase to inark inversions, the effects of genotype frequency, larval density, and culture conditions on fitness were assessed. The proportions of genotypes among egg-laying females were varied, thus changing the expected proportions of progeny produced in the absence of fecundity or viability selection. The genotypes of progeny were determined by electrophoresis and comparisons of the ratio of the numbers of the different genotypes produced to the expected ratio was used to evaluate fitness relationships. Fitness relationships were dependent on genotype frequency, larval density, and culture conditions. Selection was either absent, directional, frequency dependent (favoring rare types), or heterotic depending on density and culture conditions. It is implied that the adaptive value of genetic variants need not be apparent in all environments, or may change with changing conditions. There is evidence for different criteria for selection in the two sexes. These results add to the evidence supporting the importance of frequency- dependent selection. It is argued that for frequency dependence to be of general importance, selection must act on genes in groups, either as an inversion or as lengths of chromosome with integrity maintained by disequilibrium.

FREQUENCY-dependent selection has been demonstrated to be effective in maintaining variability in experimental populations of Drosophila (EHRMAN 1966; KOJIMA1971), Tribolium (SINNOCK1970), Mormoniella (GRANT,SNY- DER and GLESSNER1974), and Poecilia (FARR1977). In Drosophila frequency dependence has been shown in mate selection (EHRMAN1969; SPIESS1970), viability (KOJIMAand TOBARI1969a, 196913; KOJIMAand YARBROUGH1967), and fecundity (ANDERSONand WATANABE1974) components of fitness. BUND- GAARD and CHRISTIANSEN( 1972) have demonstrated frequency dependence in mate selection and viability in the same system. Studies of the viability component of fitness in Drosophila have demonstrated frequency dependence for frequency classes defined by inversion karyotypes (KOJIMA and TOBARI1969b), fourth chromosomes (BUNDGAARDand CHRIS- TIANSEN 1972), and polymorphisms marked by allozymes at the ADH (KOJIMA and TOBARI1969a) and at theesterase-6 (YARBROUGHand KOJIMA1967; KOJIMA and YARBROUGH1967) loci of D. melanogaster. The frequency dependence of

Genetm 88: 357-366 February, 1978. 358 M. H. GROMKO AND R. C. RICHMOND fitness values has been clearly shown to depend on density (KOJIMAand HUANG 1972; NASSAR,MUHS and COOK1973). A mechanism for its operation among larvae has been proposed that involves a partitioning of the environment by individuals of different genotypes (HUANG,SINGH and KOJIMA1974). The term frequency-dependent selection is used here to refer to a type of balancing selection (GROMKO1977). In a system out of genic equilibrium, the operation of frequency-dependent selection defined in this way is characterized by large differences in fitnesses between the common and rare genotypes, the latter having the highest fitness. As the rare type becomes more common. fitness differences among the genotypes lessen until, at equilibrium, the genotypes are equally fit. Since there are no fitness differences among genotypes at equilibrium, the population has no genetic load. The importance of this conclusion cannot be evaluated, however. until the generality of this mode of selection and its capabil- ity of maintaining large amounts of variability have been discovered. In the experiments to be described, the possibility that frequency-dependent selection plays a role in maintaining an inversion polymorphism in Drosophila paulistorum has been investigated. The results show that both frequency-dependent and heterotic selection are important in maintaining this polymorphism in experimental populations.

MATERIALS AND METHODS A two year old cage population of D. pzulistorum (cage IS-18 of POWELLand RICHMOND 1974) was used as the starting material for these experiments. The population was at apparent gene-frequency equilibrium for alleles at the tetrazolium oxidase (To) locus. To is a sex-linked locus with two codominant electrophoretic alleles, designated slow (S) and fast (F) (RICHMOND 1972). The cage population was segregating at approximately 60 io 65 percent of the slow allele. Eleven lines homozygous for the fast and twelve lines homozygous for the slow allozymes were isolated from the cage population by Fair matings and sib matings. Lines carrying the same allele were combined to make two homozygous stock populations from which virgin flies were collected to begin experiments. Examination of the salivary gland chromosomes of several female larvae produced from each of eight reciprocal crosses between the fast and slow lines all showed inversion heterozygosity in the right arm of the X chromosome. The intersion seen is apparently that described by DOBZHANSKYand PAVLOSKY(1962) and drawn in their Figure 7. That inversion hcterozygosity was seen in all of the preparations examined suggests that one gene arrangement was associated with the To slow allele stock and the other with the To fast allele stock. If each of the eight crosses made sampled a minimum of three X chromosomes from the two homozygous lines, then the probability that all eight crosses would show inversion lieterozygosity is less than 0.05 (= 0.8824) if the inversion and To alleles are associated 88 percent of the time. A consideration of the origin of the founder cage population also leads to this conclusion: the cage from which lines were isolated was initiated with the progeny of only two isofemale lines. This cage then is likely to have contained a maximum of six X chromosomes. Thus the association between the To alleles and gene arrangements is not necessarily surprising (POWELLand RICHMOND1974). Virgin flies one to three days old were combined in mass matings in which the To genotypes of flies of each sex were known. Four different types of mass matings were made (FF x F, FF x S, SS x F, SS x S), each having only one type of hemizygous male and one type of homozygous female. After three days females were separated from males and held for one additional day. Females from the various matings were then combined, without etherization, in known proportions and left to lay eggs in bottles of standard cornnieal-molasses medium. SELECTIOhT IN A POLYMORPHIC SYSTEM 359

TABLE 1 Scheme for uarying proportions of parental females and the expected genotypic ratios among progeny, assuming no selection

% S input Genotype of input females and mates ss x s FF X S SS X F FF X F 85 75 10 10 5 65 55 10 10 25 35 25 10 10 55 15 5 10 10 75

Zygotic input and expected genotypic ratios among progeny if each female lays 2x eggs. (Le ,z female eggs and x male eggs). % S input Females Males SS SF FF S F 85 751 20x 5x 85x 152 65 552 202 252 65x 352 35 252 202 55x 352 65x 15 52 20x 752 152 852

These mated females comprised the input of the experiment and young imagoes hatching from the culture bottles the output. The To genotypes of output flies were determined by electrophoresis, using the techniques of RICHMOND(1972). Selection in either fecundity or viability will be evident in a comparison of the genotype frequencies of flies eclosing with the frequencies expected on the basis of no selection. Three parameters were varied: (3 ) proportions of premated females, (2) larval density, and (3) culture conditions. Each experimental series consisted of four cultures differing in the proportions of premated females and thus in the expected ratios of eclosing progeny. These four input frequency conditions, represented as 85, 65, 35, and 15 percent of the S allele, and the ratios of progeny genotypes expected undcr conditions of no fecundity and no viability selection are given in Table 1. Larval density was varied by allowing the females in each series to lay first for four days, producing a “crowded larvae” series. Females were then transferred to fresh culture bottles for one day to produce an “uncrowded larvae” series. Culture conditions were varied with respect to the kind and extent of microbial growth. “Clean” cultures were maintained by putting 100 males (50 F and 50 S) into the bottles immediately after the egg-laying females were removed. These males were removed the day before the F, progeny were first expected to eclose and made no genetic contribution to the experiment. “Bacterially contaminated” cultures invariably resulted when these males were not introduced. Media in these bottles became very dark in color and dried up by the time eclosion was expected. Few or no flies eclosed in nine of ten trials maintained in this way. Media innoculated with excess yeast produced the third type of culture condition. Two series of experiments have been comFleted with clean media conditions, while there have been one of each of the contaminated series. Samples of the media from contaminated cultures were struck for isolation on a complex peptone-yeast extract agar medium, and isolates were gram stained. The bacterial contaminant present most abundantly was Bacillus. Both the bacterial and yeast contaminants were present in “clean” conditions, but at much lower levels.

RESULTS Although neutrality, directional selection, and balancing selection can be dis- tinguished in an analysis of gene (or inversion) frequencies, frequency dependence and heterosis cannot (AYALAand CAMPBELL1974; GROMKO1977). An 360 M. H. GROMKO AND R. C. RICHMOND analysis of genotype frequencies is necessary for this. The number of flies of each genotype eclosing in each culture of the eight series is given in Table 2. The fit of these numbers to the expected ratios of Table 1 have been tested in the follow- ing manner. Taking the data for female progeny from the first replica of the clean conditions, larvae crowded, as an example, the fit of the observed output at the 85 percent frequency conditions (46: 7) to the expected ratio of 15: 1 gives x;~)= 4.39, p < 0.05. The observed numbers deviate significantly from the expected ratio and do so because there are inore FF individuals (or fewer SS) than expected. Thus, relative to one another, the fitness of FF is greater than the fitness of SS. This result is summarized in Table 3A as “FF>SS*”. The relative fitnesses of these genotypes compared at the input frequency of 65 percent are not significantly different: the observed numbers (74:26) fit the expected ratio (11 : 5; xr,) = 1.28, p > 0.25), and this result is summarized as “N.S.”. Proceeding to the input frequencies of 35 and 15 percent S, the observed numbers of 20: 22 and 24: 30 deviate from the expected ratios of 5: 11 and 1: 15 with xi,) = 5.25 (p< 0.05) and x:,) = 134.20 (p< 0.005), respectively. Here the fitness relationship has reversed, however, and these results are summarized as “SS>FF*” and “SS>FF* * *”. These four fitness relationships indicate frequency- dependent selection: both homozygous types, when rare, have a higher fitness relative to the other. This frequency dependence could be attributed to selection

TABLE 2

The number of progeny of each genotype eclosing in each of the eight experimental series

Uncrowded Crowded Input %S SS SF FF S F SS SF FF S F Clean, replica 1 85 115 14 1 90 8 46 28 7 66 15 65 67 11 14 58 11 74 17 26 78 26 35 27 14 33 25 19 20 36 22 42 30 15 9 15 43 20 45 24 14 30 7 26 Clean, replica 2 85 64 8 5 55 16 75 12 1 64 7 65 47 30 19 68 53 24 44 17 37 58 35 25 12 37 52 65 24 39 20 53 25 15 7 24 84 23 84 12 9 72 20 36 Bacterial contamination 85 43 2 12 37 14 85 116 23 118 86 65 67 20 33 55 26 133 76 22 221 89 35 24 27 31 14 10 39 31 24 125 57 15 17 28 61 25 47 74 69 35 104. 54 Yeast contamination 85 16 130 38 29 181 0 65 8163 65 7 34 4 20 31 6 39 0643 35 7 44 19 38 39 4 12 11 3 26 15 1 81 28 75 38 0 33 13 26 18 SELECTION IN A POLYMORPHIC SYSTEM 361

**** *a *a xxxx*E *E *++* AAAA zzzz %E%% *** *** 362 M. H. GROMKO AND R. C. RICHMOND acting on either fecundity or viability. The equivalence of fitnesses at the input frequency of 65 percent S corresponds to the equilibrium frequency observed in the original cage population, (POWELLand RICHMOND1974). The fit of the ratio of the number of heterozygous and the number of homozygous (FFf SS) females to the expected ratio of 1:4 was tested. In the same example, the numbers 28:53 do not fit the expected ratio (xyl) = 10.74, p < 0.005). due to an excess of heterozygotes. This is summarized as “SF>Hm” in Table 3B, where Hm stands for the pooled homozygotes. Similarly, there is an excess of heterozygotes at the input frequency of 35 percent S, but not at the other two input frequencies. A heterozygote excess can be interpreted in two ways: it could be produced by a heterozygote advantage among female larvae, or it could be produced by a fecundity effect (possibly frequency dependent) of one of the two homozygous female types producing more eggs than the other. If fecundity were involved in this way, one would expect to see a correlated increase in one of the two homozygous female types. Thus, a heterozygote excess in the absence of a deviation from the expected ratio between the two homozygous female types could be interpreted as a heterozygote advantage among female larvae. The absence of heterozygote excess at the input frequency of 15 percent S where there is a large fitness difference between homozygotes (Table 3A and 3B) suggests that frequency-dependent selection among larvae predominates over any heterozygote advantage. The final step in the analysis of this series is a test of the fit of the observed numbers of male genotypes to their expected ratios (Table 3C). Again, as an example, refer to clean medium, replica 1, crowded conditions. Here, only at the input frequency of 35 percent S is there a deviation of the observed numbers (42:30) from the expected ratio (7: 13; = 17.23, p < 0.005). In Table 3, the fitness relationships for Replica 1 of the clean series with larvae uncrowded show a consistent excess of SS and S genotypes, indicating directional selection. There is no indication of heterozygote excess. In the crowded series, as discussed in detail above, selection among females is clearly frequency dependent, while selection among males shows no significant trend. Replica two of the clean conditions shows overall similarities to the first, with several differences in detail. In uncrowded conditions, no consistent deviations from expecta- tions are observed. With crowding, there is a tendency towards frequency- dependent selection, particularly among males, but not nearly so strongly as among females in the first replica. Taken together, the results in the clean, uncrowded conditions show predominantly directional selection or no selection. With crowding, there is a tendency towards frequency-dependence. There is also a slight tendency for heterozygote excess, which could be due either to heterozygote advantage or to a fecundity effect. Although fecundity and viability are confounded, viability selection has probably been more important than fecundity in producing a heterozygote excess such as just described, which appears in only one of two densities within any one condition. Density differences are differences in larval density; the total number of egg-laying females was held constant. The specific differences between the replicas of clean medium are difficult to SELECTION IN A POLYMORPHIC SYSTEM 363 explain. Since these two replicates were run at different times, it is possible that deviations in media conditions could account for the differences between replicas. Our observations that experimentally induced alterations in media (see below) affect fitness relationships support this hypothesis and underscore the sensi- tivity of this system to media conditions. In the series run in medium contaminated by bacteria, selection is clearly and consistently frequency dependent. Both homozygous female genotypes and the two hemizygous male genotypes are in excess when rare, compared to the common type. In uncrowded conditions, there is an excess of heterozygotes at only one frequency, suggesting that frequency-dependent selection is largely or solely the cause of the observed changes. With crowding, selection becomes much more strongly balancing. The output gene frequency is close to 65% S at all input frequencies. There is also a large excess of heterozygous females at all input frequencies. This could be due either to an intensification of a frequency- dependent fecundity effect (which does not seem likely because of the constant parental female density) or tc\ an overlay of heterozygote advantage among larvae on the basic frequency-dependent pattern. The situation in the yeast-contaminated series is markedly different. There were no striking differences in the relative fitness of genotypes due to crowding. Homozygous females, with one exception, showed no significant deviations from the expected ratios. Frequency-dependen t selection in either fecundity or viability was not, therefore, involved in determining the relative output of the female genotypes. However, there was an excess of heterozygous females at all frequencies in both crowded and uncrowded cultures. Because there was no difference between the fitness of homozygous female genotypes, the heterozygote excess must be due to heterozygote larval advantage and not to a fecundity effect. Importantly, selection among males shows a very different pattern from that found among females: selection strongly favors rare males. Males, hemizygous for the inversion, and most of the X chromosome as well, would be at a serious disadvantage in these conditions if selection were strictly heterotic, making the sex ratio deviate from 1: 1. Approximately equal numbers of males and females were produced in these conditions, however, (Table 2) indicating different criteria for selection in the two sexes.

DISCUSSION The genotype frequencies of flies produced in these experiments have been affected by their frequency, the larval densities and the condition of the culture medium. Selection has been absent, directional, frequency dependent, and heterotic, depending on the combination of the above-mentioned factors. Larval crowding, within any one culture condition (except yeast contamination) tends to emphasize frequency-dependent selection, a result that is in agreement with previous work (KOJIMA and HUANG1972; NASSER,MUHS and COOK1973). Bacterial growth in the culture medium also produces frequency-dependent selection. Contamination of the culture medium by excess yeast, however, results 364 M. H. GROMKO AND R. C. RICHMOND in heterozygote advantage among female larvae regardless of genotype frequencies, while hemizygous males show strong frequency-dependent fitnesses. That relative fitnesses are extremely sensitive to physical and biotic properties of the culture medium has been demonstrated by other workers ( DA CUNHA195 1 ; DOBZHANSKYand SPASSKY1954). The observation that the mode of selection can shift depending on conditions in the environment suggests that genetic variants need not be selected for in every generation in order for them to have adaptive value. The detection of selective differences may depend on the discovery of appropriate environmental conditions (WILLS,PHELPS and FERGUSON1975). Both genotype frequency and environment are important in determining the action of selection in this system. These two factors may be related to one another in a fundamental way. Frequency dependence can be a special case of environment dependent or “multiple niche” polymorphism ( LEVENE1953; POWELL 1971; HEDRICK, GINEVAN and EWING1976). Conditioned media studies (HUANG, SINGHand KOJIMA1974; KOJIMAand HUANG1972) suggest the modeling of frequency-dependent selection by differential utilization of the environment by different larval genotypes. If resource utilization were thus partitioned, selection would favor rare types because of limited competition in their niche. Frequency- dependent selection would drive the genotype frequency to a point where the frequencies of the alternative genotypes would be the same as the frequency of the separate niches. The role of density in manifesting frequency dependence in such a situation has been discussed in detail by KOJIMAand HUANG(1972). The results presented here suggest that stresses other than density can be effective in producing frequency-dependent selection. In terms of the above model, the addition of stressful conditions to a culture might increase competition for resources or even provide a novel partitioning of resources not present before. Furthermore, the addition of a stress to the environment can change the pattern of fitnesses from neutrality or frequency dependence to one with consistent heterozygote advantage. One important objection to frequency dependence as a general mechanism is that it would require the environment to be subdivided in as many ways as there are polymorphisms maintained by frequency-dependent selection (LEWONTIN 1974). Moreover, it is difficult to imagine how the confusion of rare and common types at many loci could be sorted out. Is an individual with 15 percent of its polymorphic loci occupied by rare alleles and the remainder by common ones seen by selection as rare or common? This objection, of course, is less important if the genetic system in question is an inversion polymorphism. The association of genes in groups may be possible even in the absence of inversions (FRANKLIN and LEWONTIN1970). If linkage disequilibrium were to result in the association of genes with related functions, it is possible that a single partitioning of environmental resources could account for the existence of several polymorphic loci. Theoretical considerations (FRANKLINand LEWONTIN1970; WILLS,CRENSHAW and VITALE1970) and empirical observations (BAKER1975; TEMPLETON,SING and BROKAW1976) suggest that selection acts on lengths of chromosome and not on individual loci as units. YAMAZAKI(1974) has approached this problem SELECTION IN A POLYMORPHIC SYSTEM 365 directly for the case of frequency dependence in a computer simulation of the evolution of linked genes. He found evolution in the system leading to associations of alleles at loci in blocks of LIP to 10 in length, with the minority alleles becoming associated in groups separately from the associations of majority alleles. Thus, the generality of the phenomenon of frequency-dependent selection may not be restricted because of the “necessity” of maintaining loci in polymorphic condition independently of one another. This study adds another genetic system to the growing list of polymorphisms that can be shown to exhibit frequency-dependent selection. The variability of fitness values here is dependent on environmental conditions, as well as on frequency and density. The mode of selection in the inversion system studied has changed from no selection to frequency dependence to heterozygote advantage, depending on culture conditions. We are very grateful to LINDAIC. JONES,for the examination of salivary gland chromosomes, and to T. KAUFMANfor identifying the inversion loop. DR. D. W. PYLEand B. J. COCHRANE critically read the manuscript and C. WILHFLMand B. LETWINprovided valuable technical assistance. We are also grateful to P. MUSSELWHITEand W. E. KLOOSfor identifying the microbial fauna associated with the cultures. North Carolina State University provided space and facilities during preparation of the manuscript. This work was supported in part by Public Health Service Genetics Training Grant No. 82 awarded to Indiana University, and in part by Grant No. GM18690 to DR. R. C. RICHMOND.

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