Polygenic mutation in Drosophila melanogmter - Semantic Scholar

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low but not high abdominal bristle number, but the interpretation is confounded by variation in ...... tations are practically additive ( CROW and SIMMONS. 1983) ...
Copyright 8 1995 by the Genetics Society of America

Polygenic mutation in Drosophila melanogmter: The Causal Relationship of Bristle Number to Fitness Sergey V. Nuzhdin, * *+J James D. Fry* and Trudy F. C. Mackay” *Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695- 7614, and +Institute of Molecular Genetics, Moscow 123182, Russia

Manuscript received May 25, 1994 Accepted for publication November 5 , 1994 ABSTRACT The association between sternopleural and abdominal bristle number and fitness inDrosophila melane gaster was determined for sublines of an initially highly inbred strain that were maintained by divergent artificial selection for 150 generations or by random mating for 180 generations. Replicate selection lines had more extreme bristle numbers than those that were maintained without artificial selection at the same censussize for approximately the same number of generations. The average fitness, estimated by a single generation of competition against a compound autosome strain, was 0.17 for lines selected for high and low abdominal bristle numbers and 0.19 for lines selected for high and low sternopleural bristle number. The average fitness of unselected lines, 0.46, was significantly higher than that of the selection lines.The fitnesses and the relationships of bristle number to fitness in progenyof all possible crosses of high X high ( H X H ) , high X low ( H X L ) and low X low ( L X L ) selection lines were examined to determine whether the observed intermediate optima were caused by direct stabilizing selection on bristle number or by apparent stabilizing selectionmediated through deleterious pleiotropic fitness effects of mutations affecting bristle number. Although bristle number was nearly additive for progeny of H X H, H X L and L X L crosses among sternopleural bristle selection lines, their mean fitnesses were not significantly different from each other, or from the mean fitness of the unselected lines, suggesting partlyor completely recessive pleiotropic fitness effects causeapparent stabilizing selection. The average fitness of the progeny of H X H abdominal bristle selection lineswas not significantly different from the fitness of unselected lines, but the mean fitness of the progeny of L X L crosses was not significantly different from that of the pure low lines. This is consistent with direct selection against low but nothigh abdominal bristle number, but the interpretation is confounded by variation in average degree of dominance for fitness (on average recessive in the high abdominal bristle selection linesand additive in the low abdominal bristle selectionlines). Neither direct stabilizing selection nor pleiotropy, therefore, can account for all the observations.

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HE mean number of sternopleural and abdominal Drosophila melanogaster varieslittle bristles of among natural populations ( KFARSEY and BARNES1970; ROFFand MOUSSEAU1987), o r over time withina single population. Yet these traits typically have high levels of genetic variance, and average bristle numbers far outside the range of the initial base population can be achieved readily in populations subjected to divergent artificial selection. T h e evolutionary force ( s ) responsible for the apparently contradictory observations staof ble means and abundant genetic variation of Drosophilabristle number have been debated for nearly 40 years. The question at the heart of the debate is, “What is the relationship between numbers of bristles and fitness?” Two contrasting relationships ofbristle number to fitness have been proposed. O n the one hand, the stability ofmeans across populationsand time has been Correspondingauthor: Trudy F. C. Mackay, Department of Genetics, Box 7614, North Carolina State University, Raleigh, NC 27695-7614. E-mail: [email protected] A-esent address: Department of Genetics, Box 7614, North Carolina State University, Raleigh, North Carolina 27695-7614.



Genetics 199 861-872 (February, 1995)

taken as evidence that the bristle traits are subjected to stabilizing natural selection, such that individuals with bristle numbers near the optimum have higher fitnesses than individuals with more extreme numbers of hairs ( MATHER 1941). On the other hand, immediate and large responses to artificial selection have been used to argue bristles are selectively neutral, with n o fitness differences between individuals over much of the phenotypic range (ROBERTSON1955) . Experiments to distinguish the two hypotheses have yielded ambiguous a n d / o r contradictory results. O n e test of the relationship of any quantitative character to fitness is to perturb the mean value of the trait from its initial value in both directions by several generations of artificial selection and then to relax artificial selection and maintain the selected linesunder competitive conditions where natural selection operates. If none of the alleles responsible for the selection response have been fixed, one should observe a return towards the initial population mean if stabilizing selection acts on thetrait. Seven independent replicates of five generations of divergent artificial selection for abdominalbristle number

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from the longestablished Kaduna base population, followed by 9-19 generations of relaxed selection under crowdedcompetitive conditions, have been reported (CLAYTON et al. 1957; LATTER and ROBERTSON 1962). The relaxed high lines retained on average 67% of the response gained under selection, and the low lines retained 76% of the initial response. Similarly, five replicate lines have been selected for 4 or 5 generations for high and low sternopleural bristle number from Kaduna and two (at the time) recently caught natural populations, followedby 19-25 generations of relaxation ( LATTER and ROBERTSON1962;SPIERS 1974). The relaxed high lines retained on average 54% of their initial response while the low lines retained 67%. These experiments, andothersdemonstratingthe relaxed selection lines retained sufficient genetic variation to respond readily to renewed artificial selection, led ROBERTSON (1967) to concludethatthe forces maintaining genetic variation for bristle number (in the laboratory) “. . . are not very strong and that the genes at a high proportion of loci must be almost neutral in their effects on fitness.” Yet we must infer from the repeated observation that some lossof selection response usually occurs, that natural selection operates on alleles of at least some of the loci responsible for variation in bristle number.Indeed, KEARSEY and BARNES (1970) inferred strong stabilizing selection acting on sternopleural bristle number from the change in phenotypic distribution andreduction of genetic variation of a population derived from a cross of lines selected for high and low bristle numbers, when grown under highly competitive population cage conditions compared to low-density, noncompetitive conditions. However, we need to know the causal relationship between bristle number (or any other quantitative trait) and fitness if we are to understand the evolution of quantitative genetic variation. In this regard an observation that individuals with intermediate phenotypes have higher fitness than more phenotypically extreme individuals is oflittle help, for there areseveral different possible genetic mechanisms that will give rise to this observation (ROBERTSON 1967). The classic model of stabilizing selection is of a causal relationship between values of the trait and fitness (“direct” stabilizing selection). “Apparent” stabilizing selection arises if selection is operating on a genetic property of loci affecting the trait unconnected to its phenotypic values, such as overdominance of alleles associated with intermediate trait values (ROBERTSON 1967; BARTON1990) or deleterious pleiotropic effects on fitness ofalleles causing extreme phenotypes (HILL andKEIGHTLEY 1988; BARTON 1990; KEICHTLEY and HILL1990;KONDRASHOV and TURELLI 1992; GAVRILETS and DE JONG 1993) . ROBERTSON (1967) argued that direct stabilizing selection is not likely a pori,because the model essentially subdivides organisms into as many compartments as there are imaginable traits, each of which causally affects fitness

through its mean phenotypic value. KEARSEY and BARNES(1970) concluded that the selection they observed was on pleiotropic effects of alleles affecting adult bristle number on larval competitive ability, because selection was at the preadultstage. An alternative and not mutually exclusive explanation is that the parental high and low selection lines used to initiate the population in which stabilizing selection was observed had different fixed recessive alleles withdeleterious fitness effects; individuals with extreme bristle numbers would havelow fitness because they are homozygous for linked deleterious alleles, whereas intermediate-scoring individuals would be heterozygous for both bristle loci and unrelated genes affecting fitness and be favored by selection. It has proven exceedingly difficult to discriminate experimentally among thevarious potential causal relationships between bristle number and fitness. LINNEY et al. (1971) reasoned that if they determined fitness of homozygous lines with different bristle numbers, they could eliminate selection on loci withdeleterious recessive fitness effects that arelinked to loci affecting bristle number as a potential explanation for the stabilizing selection observed by KEARSEY and BARNES(1970). They found that homozygous lines with extreme sternopleural bristle numbers had reduced competitive viability compared with those with intermediate scores and again concluded apparent stabilizing selection acted on alleles affecting bristle number through their deleterious pleiotropic effects on larval competition. ROBERTSON ( 1967, 1970) constructed chromosome substitution lines from high and low sternopleural bristle number selection lines of genotype L1 L2 H3 L4 and HI Hz L3 H4, where H and L refer to chromosomes from the high and low lines, respectively, and the subscripts refer to the four Drosophila chromosomes. Crossing these lines to eitherthe high or low selection line yielded four synthetic populations: HI Hz H3/LB H4; L, L2 H3/L3 L4; H 1 / b H J L 2H3 H4/L4 and H I / L I H2/ L2 L3 H4/ L4. These populations were allowed to segregate and evolve in population cages for several years. The prediction was that if natural selection acted directly on bristle number, the mean bristle number in these lines wouldevolvetoward theintermediate optimum. The mean bristle number did not change significantly. ROBERTSON ( 1967,1970) concludedfrom this that bristle number is a neutral trait unrelated to fitness. However, these results could have occurred also if there was strong selection for heterozygosity at bristle number loci or there was selection for heterozygosity of deleterious recessive alleles linked to bristle loci. To determine whether hitchhiking of deleterious recessive alleles could have accounted for the behavior of ROBERTSON’S synthetic populations, SPIERS(1974) extracted different isogenic third chromosomes of genotype HI Hz H3 H4 and HI HzL3 H4 from the original chromosome substitution lines andcompetedthem

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locus in each line (e.g., each line could be fixed for a new mutation affecting bristle number, but the locus at which the mutation occurred would be different for every line) . Determining thefitness and bristle number of isogenic lines homozygous for different bristle loci and for heterozygous genotypes produced by crossing the homozygous lines should enable inference of the causal relationship of bristle number to fitness. The following outcomes are possible, assuming bristle number is additive.

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FIGURE 1.-Relationship between sternopleural bristle number and fitness from the data of SPIERS(1974). Each point represents a homozygous third chromosome line, and fitness is estimated by the frequency of wild-type third chromosomes after 3 generations of competition against the third chromosome balancerTM2 ( 0 ) or 14 generations of competition against TM3 ( ) .

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against two third chromosome balancers in separate experiments. The equilibrium frequency of the H or L third chromosome is an overall measure of its fitness relative to that of the balancerchromosome ( SVED 1971, 1975). Individuals of genotype HI Hz L3 H4 had wild-type (an average of 17 sternopleural bristles) bristle numbers whereas the HI Hz H3 H4 genotypes had on average 38 sternopleural bristles; under the direct stabilizing selection hypothesis the L3 chromosomes would be expected to have higher fitness than the H3 chromosomes. There was no apparent association between bristle numberand fitness (Figure l ) , from which SPIERS ( 1974) concluded thatbristle number is a neutral character unrelated to fitness. However, neither these observations nor those of ROBERTSON exclude the possibilities that alleles affecting bristle number have pleiotropic deleterious effects on fitness or that there is classical overdominance of bristle effects. Contradictory interpretations of the causal relationship of bristle number to fitness from the experiments described above stem from confoundingeffects of background genotype, and because the dominance propertiesof possible fitness effects and the correlation of effects on fitness with effects on bristle number for alleles at loci affecting bristle number are unknown. An ideal experimental test of the causal relationship of bristle number (or any other quantitative trait) to fitness would require a series of homozygous lines that are genetically identical except forone different bristle

1. If bristle number is a selectively neutral trait, there will be no association between bristle number and fitness for homozygous and heterozygous genotypes. Furthermore, theaverage fitness of the homozygotes and heterozygotes will be the same. 2. If there is directional natural selection for bristle number, then for both homozygous and heterozygous genotypes there will be a monotonic relationship between bristle number and fitness, such that bristle number phenotypes at one end of the range are most fit whereas those at the otherend are least fit. 3. If stabilizing selection acts directly on bristle number, then thefitness of individuals with extreme bristle number phenotypes will be less than those with intermediate bristle numbers, for both homozygous and heterozygous genotypes. 4. If there is classical overdominance operatingon bristle number loci, then there will be no association between bristle number and fitness of homozygous genotypes, but thefitness of heterozygous genotypes will be greater than that of homozygous genotypes. 5. If bristle loci have deleterious pleiotropic effects on fitness, then homozygous genotypes will show an intermediateoptimum if there is acorrelation ( p , HILLand KEICHTLEY 1988; KEICHTLEY and HIL.L 1990) between the magnitude of bristle effects and decline in fitness, or show no association of bristle and Tunumber and fitness if p = 0 ( KONDRASHOV RELLI 1992). The predicted association of bristle number and fitness for heterozygous bristle loci under the pleiotropic model depends on the degree of dominance of fitness effects of alleles affecting bristle number and on the magnitude of the correlation between bristle effects and fitness effects. If fitness effects are fully or partly recessive, all heterozygous genotypes willhave greater fitness thanthe homozygotes. For fully recessivefitness effects, there will be no association between bristle number and fitness because in this case fitness effects will fully complement regardless of the bristle number phenotype. If fitness effects are partly recessive, there may be an association between deviation of bristle number from the optimum and fitness if p = 0. If fitness effects are additive or partly dominant, theheterozygotes will have the same or lower fitness than the

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homozygous lines, because they will harbor more deleterious mutations ( KONDRASHOVand TURELLI 1992) ; there may or may not be a furtherreduction of fitness ofindividualswith extreme bristle numbers depending on whether or not fitness and bristle effects arecorrelated.The onlyambiguityin this scheme is that it is not possible to distinguish selective neutrality from the case of pleiotropy where p = 0 and fitness is strictly additive. We have derived mutation accumulation lines of Drosophila from a common highly inbred base population. One set of lines was maintained for -150 generations by replicated divergent artificial selection foreither sternopleural or abdominal bristle number ( MACKAY et al. 1994) while the other set was maintained for -180 generations at the same census population size without artificial selection ( MACKAY et al. 1992a, 1995). Thus these lines partially satisfy the criteria for determining the causal relationship of bristle number to fitness in that they were initiallyisogenic and have different mean bristle numbers from new mutations: the selected lines have diverged by an average of 12.9 abdominal and 9.9 sternopleural bristles from the base population average, whereas the average bristle numbers of the unselected lines did not change from the base population values. However, they vary by more than a single bristle mutation (FRYet al. 1995) and have potentially accumulated deleterious background mutations unrelated to bristle number. We estimated fitness by competition against a compound autosome strain ( HARTLandJUNGEN 1979) for the selected and unselected lines and for progeny resulting from all possible crossesof high X high, high X low and low X low replicate selection lines for each trait, to infer the causal relationship of bristle number to fitness. MATERIALS AND METHODS

Drosophila strains: Mutation accumulation lines were derived from a highly inbred Harwich base population. Unselected lineswere maintained by mass matings of 10 randomly chosen individuals of each sex per generation ( MACKAY et al. 1992a) and selected lines by divergent artificial selection for abdominal or sternopleural bristle number at thesame population size of10 selected pairs of parents per generation (from 40 of each sex scored to generation 64; from 20 of each sex scored subsequently) with three replicate selection lines for each trait/direction of selection ( MACKAY et al. 1994). The fitness estimates described below were made for a subsetof 10 of the 20 unselected lines described in MACKAY et al. ( 1995) at generation 182 of mutation accumulation and for all 12 selection lines between generations 148 and 153. Selection was relaxed for two to four generations before the fitness measurements, to obtain sufficient flies for theassays. All lines used had identical sites of insertion of the retrotransposon elements 297 and mdg3 (NUZHDIN andMACKAY 1994), so initial polymorphism of the base population Hanvich inbred line and/or contamination by unrelated flies can be ruled out as sources of variation in fitness or bristle number. At the time of the fitness assays, the unselected strains used had an

average of 15.4 sternopleural bristles, compared with 22.6 and 12.7 for the high and low sternopleural bristle selection lines, respectively. The mean abdominal bristle numbers in the unselected lines, and in the high and low abdominal bristle selection lines were, respectively, 15.3, 17.6 and 4.7. Thecompound autosome ( C A ) strain C(2L)RM, dp; C(2R)RMpxwas used as a competitor for themutation accumulation lines in the fitness assays. In this strain the two left arms of chromosome 2 are joined toa common centromere, as are the two right arms. Progeny resulting from crosses of this strain to a chromosomally normal strain are inviable. Fitness assays: The competitive index (CI) technique was used to estimate fitness (KNIGHT and ROBERTSON 1957; HARTL andJUNCEN 1979). Toestimate fitness using this method, unmated males and virgin females of the strain to be tested are put together in a culture vial with males and virgin females of the marked CA strain. The competitive index is the ratio of the number of offspring of the tested strain to the total number of progeny; because the CA and tested strains are effectively reproductively isolated there are no interstrain cross progeny. The initial proportion of CA and tested flies is chosen to give an average CI for all the strains tested of -0.5, to maximize the power to detect differences in fitness among the tested strains. The CI weights the fitness components of male mating ability (male fertility, M ) , female fecundity, F and viability, V as M X F X V (KNIGHT and ROBERTSON 1957) and correlates very well with a composite fitness index constructed fromindividual fitness components ( HAWER and HARTL 1983). Fitness was estimated for 94 different genotypes, comprising the mutation accumulation lines and their F1 progeny: a set of 36 lines resultingfrom a full diallel cross of allreplicates of high and low sternopleural bristle selection lines, an analogous set of 36 lines from the diallel cross of high and low abdominal bristle selection lines, 10 unselected lines and 12 lines from a full diallel cross of a subset of 4 of the unselected lines. Because homozygous genotypes segregate in the progeny of heterozygous genotypes, heterozygous fitness may be underestimated, by an unknown amount that depends on the strength of viability selection against homozygous genotypes. All cultures were maintained in shell vials with -10 ml cornmeal-agar-molasses medium at 25". To produce flies for the fitness assays reared under standardized conditions, five males and five virgin females from the mutation accumulation lines to be tested were placed in a culture vial for 8 days. Flies from the CA strain were reared contemporaneously at the same density. Virgin females and unmated males from themutation accumulation lines or their F1 hybrids, and from the CA strain, were collected for 7 days after the first flies eclosed from the low-density cultures. On the 8th day from the first eclosion, 10 replicate vials were set up for each of the genotypes to be tested, each with four males and four females of the tested genotype and 10 pairs of CA flies. Males ofboth strains were kept together for 5-9 hr in vials without food to recover from ether anesthesia before introducing them to the females. Flies were removed from the vials after 8 days, and the progeny were counted 9-10 days later. Bristle number: Bristle number was scored on 20 males and 20 females from each of the 94 genotypes tested in the fitness assay. F1 individuals from crosses of mutation accumulation lines were sampled from the 40 individuals of each sex used to initiate the 10 replicate CA competition vials. Bristle numbers for the pure selected and unselected lines were o b tained from flies at the appropriate generation of mutation accumulation. The sum of the number of sternopleural bristles on the left and right sternopleural plates was recorded for all crosses of the three high and three low sternopleural bristle selection lines, and for all unselected lines and their

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Selection on Polygenic Mutations crosses. The number of abdominal bristles on the most posterior abdominal sternite (segment six of females, five ofmales) was recorded for all crosses of the three high and three low abdominal bristle selection lines, and also for all unselected lines and their crosses. Statistical analyses: Mean fitness of lines was regressed on their deviations and squared deviations from mean bristle number. Estimates of the linear and quadratic effects and Type I11 (simultaneous) sums of squares were calculated using the Proc GLM procedure in SAS (SAS INSTITUTEINC. 1988). Simple t-tests or one-way analyses of variance of line means were usedto test significanceof differences in overall means of two or more groups of lines, respectively.

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CIS and bristle numbers were estimated for a total of 94 genotypes: ( 1) 10 unselected lines at generation 182 of mutation accumulation; ( 2 ) the F1 progeny from all possible crosses among a subset of four of the unselected lines ( 12 genotypes) ; (3) replicate lines selected for -150 generations from an inbred base population for high ( H ) and low ( L ) sternopleural bristle number ( 6 lines) and F1 progeny of all possible H X H, H X L and L X L crosses among the replicate sternopleural bristle selection lines (30 genotypes) ;and ( 4 ) replicate lines selected for high and low abdominal bristle number ( 6 lines) and all possible crosses among the high and low abdominal bristle selection lines (30 genotypes) . Mean CISand bristle numbers aregiven for each of the 94 genotypes in Appendixes A-D and plotted in Figure 2. CIS and bristle numbers averaged overall replicates of unselected and selected lines and progeny of crosses are summarized in Table 1. Selection onbristle number: The average sternopleural and abdominal bristle numbers of the unselected lines were15.4 and 15.3,respectively (Table 1 ) . The mean bristle numbers for these traits over all replicates of the unselected lines has remained stable over 180 generations of mutation accumulation (MACKAY et al. 1992a, 1995) , so we have assumed the grand mean of these lines is near the optimum. Responses to selection for both sternopleural and abdominal bristle number were asymmetrical. The average deviation from the assumed optimum of the high and low sternopleural bris tle number selection lines was 6.6 and -2.9 sternopleural bristles, and for the high and low abdominal bristle selection lines was2.9 and -10.2 abdominal bristles. The average fitness (CI) of the unselected lines (0.46) was significantly higher than the average of linesselected for high and low sternopleural bristle number (0.19, tI4 = 3.86; 0.001 < P < 0.01) and of lines selected for high and low abdominal bristle number (0.17, tI4 = 3.76; 0.001 < P < 0.01 ) . Formal analyses ofthe regression of competitive index on deviation of bristle number from the assumed optimum for the two traits in selected and unselected lines showed that in both cases the linear regression coefficient was not significant (-0.014 2 0.017 for sternopleural bristles, and 0.020 2 0.018 for

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FIGURE2.-Mean bristle numbers and competitive indices of mutation accumulation lines derived from a common inbred base population, and of crosses among them. 0 and 0, unselected lines and F1 progeny of crosses among them, respectively; A and A, high selection linesand crosses, V and V,low selection lines and crosses; 0, progeny derived from high X low selection line crosses.Data for sternopleural bristle number are shown at top and for abdominal bristle number at bottom.

abdominal bristles). However, quadratic regression coefficientsweresignificant (-0.0051 2 0.0021 for sternopleural bristles and -0.0069 -+ 0.0029 for abdominal bristles), indicating lines with extreme bristle number had reduced fitness compared with lines with bristle numbers near the assumed optimum. Because the selected and unselected lines were ini-

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TABLE 1 Mean competitive indices and sternopleural (ST) and abdominal (AB) bristle numbers of unselected and selected mutation accumulation lines and progenyof crosses among them

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7,704 9,762 1,848 1,653 5,157 15,680 5,527 1,816 1,846 4,761 11,371 3,576

0.460 2 0.050 0.499 2 0.019 0.200 2 0.043 0.176 5 0.061 0.350 2 0.072 0.427 t 0.016 0.392 2 0.020 0.235 5 0.059 0.107 2 0.079 0.506 2 0.061 0.328 t 0.025 0.178 t 0.021

15.4 2 0.16 15.0 15.5 5 0.10 22.0 t 2.10 12.5 t 0.93 21.6 5 0.90 16.9 t 0.25 13.8 5 0.11

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18.2 5 0.41 5.1 2 1.82 18.4 2 0.24 12.8 2 0.49 6.6 t 0.82

Values are means 2 SE. SEs are empirical, computed from variance among replicate lines. U S and USX, unselected mutation accumulation lines and progeny of crosses between them, respectively; H, high; L, low; S, sternopleural; andA, abdominal bristle selection lines and progeny of all possible crosses among them; N, number of replicate lines; Total, total numberof flies scored per genotype. tially derived from the same highly inbred base population and were maintained at the same census population size for roughly the same number of generations at the time of the fitness measurements, we can assume as a first approximation that theaverage level ofdeleterious mutations at loci that do not affect bristle number is roughly equivalent in the two groups of lines. The major difference between the selected and unselected lines, therefore, is that the former have accumulated mutations with large net effects on bristle number, and the observation of significantly reduced fitness of these lines compared with unselected lines can be taken as evidence for selection operating against mutations causing deviation in bristle number from the optimum. Causal relationship of sternopleural bristle number to fitness: The mean sternopleural bristle numbers of offspring of all possible H X H and L X L crosses between replicate sternopleural bristle selection lines (21.6 and 13.8, respectively) were not significantly different from the means of the pure high and low line replicates (Table 1) . The average bristle number of progeny from H X L crosses (16.9) was significantly different from the bristle numbers of H X H and L X L cross progeny. From these data we conclude new mutations affecting bristle number in the selected lines act on average nearly additively on the selected trait, with partial dominance oflow alleles over high (see also FRYet al. 1995) . The average competitive indices of the progeny from crosses of H X H, L X L and H X L replicate selection lines (0.35, 0.39 and 0.43 respectively; Table 1) were not significantly different from each other ( F2,27= 1.49, P > 0.05). However, the regression analysis of fitness on deviation of bristle number from the assumed optimum for these lines showed the linear regression coefficient was not significant (0.0011 2 0.0063) but that

the quadratic regression coefficient was highly significant (-0.0049 2 0.0014).The significant quadratic regression is due to the low fitness of progeny from reciprocal crosses of high selection replicates 2 and 3 (see Appendix C ) ; the quadratic regression coefficient when progeny from reciprocal crosses ofthese lines are excluded from the analysis is nonsignificant (0.0001 ? 0.0024). Furthermore, the average CI of all inter-line crosses (0.40) was significantly higher than that of the pure high and low sternopleural bristle selection lines (0.19; b4 = 5.06, P < 0.001) but was not significantly different from the mean fitness of the 10 unselected lines (0.46; bs = 1.31, P > 0 . 0 5 ) . These data indicate fitness effects of alleles affecting bristle number were not additive nor dominant; if they were, fitnesses of all heterozygous genotypes would be equal to or less than that of the pure lines. Neither were fitness effects completelyrecessive. The average Cl of F1 progeny of crossesbetween unselected lines (0.50) was significantly greater than the average CI of offspring from all crosses between high and low selection lines (0.40; tm = 3.07, 0.001 < P < 0.01) ; all heterozygotes had approximately equal fitness, with the two exceptions noted above, with partially recessive fitness effects. Our data are thus largely consistent with the cause of the observed selection against nonoptimal sternopleural bristle numbers being partly recessive,deleterious pleiotropic effects on fitness of alleles affecting bristle number. However, the low fitness of reciprocal crosses of high selection replicates 2 and 3 could have several explanations: direct selection against high bristle number, a high pleiotropic correlation between bristle and fitness effects of mutations in these lines, or allelism or epistasis ofbristle mutations in the two lines. Causal relationship of abdominal bristle number to

Selection Mutations on Polygenic fitness: The mean abdominal bristle number of progeny of crossesamong replicate high ( 18.4) or low (6.6) selection lines were not significantly different from the average of the pure lines. Progeny of H X L selection line crosses averaged 12.8 abdominal bristles, as would be expected foradditive bristle effects (Table 1) . However, the patternof heterozygous fitnesses observed did not fit any of the models outlined above. The mean competitive indices of progeny of H X H, H X L and L X L replicate line crosses were, respectively:0.51, 0.33 and 0.18; the difference in mean CIS was highly significant ( F2,27= 14.1, P < 0.001 ) . The linear regression of CIon deviations of abdominal bristle number of cross progeny from the optimum was highly significant (0.028 ? 0.0043) but the quadratic regression was not significant (0.0006 ? 0.0009) . Thus we have the paradoxical situation in which comparison of fitnesses of pure selected and unselected lines shows clear evidence of an intermediate optimum for bristle number, but comparison of fitnesses of heterozygous lines with low, intermediate and high bristle numbers points to selection against low bristle number. The reason for this paradox appears to be violation of our tacit assumption that theaverage degree of dominance for fitness of mutations affecting bristle number would be the same for mutations increasing and decreasing the value of the trait. For the case ofmutations affecting abdominal bristle number, those increasing the trait value in these selection lines had completely recessive effects on fitness: the average fitness of H X H crosses (0.51 ) was significantly greater than theaverage of the pure high lines (0.24; t7 = 2.80, 0.01 < P < 0.05) and not significantly different from the average fitness of unselected lines (0.46; t14 = 0.57, P > 0.05) or crosses among unselected lines (0.50; tI6 = 0.13, P > 0.05). Mutations in the low selection lines, on the other hand, had on average nearly additive fitness effects: there was no significant difference between the average fitness of the pure low lines ( 0.11) and of the L X L crosses (0.18; t7 = 1.18, P > 0.05). Fitnessof the H X L cross progeny was intermediate between that of H X H and L X L cross progeny, as would be expected from a mixture of additive and recessive fitness effects of mutations affecting bristle number. Thus the heterozygous fitness data of the abdominal bristle selection lines are consistent with the causal relationship between bristle number and fitness being one in which deleterious pleiotropic sideeffects on fitness of mutations affecting bristle number cause apparent stabilizing selection in homozygous lines, with variable degrees of dominance for fitness of bristle mutations. The heterozygous fitness data of the abdominal bristle selection lines are also consistent with directional selection favoring high bristle number, but this hypothesis does not explain the apparent stabilizing selection on pure lines, nor the stability of mean abdominal bristle

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number of the group of unselected lines over 180 generations of mutation accumulation. DISCUSSION

What is the causal relationship of sternopleural and abdominal bristle number in Drosophila to reproductive fitness? We have tried to infer this relationship for new spontaneous mutations by ( 1 ) comparing bristle numbers and fitness of lines derived from a common inbred base population, thatwere either unselected for 180 generations or selected for increased and decreased numbers of bristles for 150 generations; and ( 2 ) determining therelationship between bristle number and fitness in progeny of all possible crosses among high and low replicate selection lines. For both bristle traits, the fitness, estimated by the competitive index method (KNIGHT and ROBERTSON1957; JUNGEN and HARTL 1979), of lines selected for increased and decreased numbers of bristles was less than that of the unselected lines. From this relationship we infer that mutations at loci affecting bristle number are notselectively neutral, nor is there directional selection for mutant alleles increasing or decreasing bristle number. Rather, these observations are consistent with selection against mutations with large net effects on bristle number. Strength of stabilizing selection: If stabilizing selection acts on bristle number, its “strength,” w2/Vp, can be estimated from the CIS and bristle numbers of the pure selected and unselected lines, assuming the noroptimal model where fitness declines as a function of the squared deviation from the optimum bristle number.ThenW(X) = W,exp(-X‘/2w2),=WO(1-X2/ 2w2) near X = 0. (Here, X is the deviation from the optimum bristle number and W, is the fitness at X = 0.) The slope of the regression of [ W ( X ) - W,] / W, on X2,forced through zero, estimates -1 /2w2. Using the mean bristle numbers and CIS of the unselected lines as the estimate of the optimum bristle number and W,, respectively,gives w 2 = 51 for sternopleural bristles and 77 for abdominal bristles. Scaling by the phenotypic variance, estimated from the mean variance in bristle number within unselected lines, givesestimates of the strength of stabilizing selection of 41 for sternopleural bristles and 25 for abdominal bristles. The estimates indicate fairly strong stabilizing selection, and are of the same magnitude as those commonly quoted for other quantitative traits in natural populations (TURELLI1984). These estimates areabout 10 times higher than those of MACKAY et al. (1995), computed from change in variance and covariance among the unselected mutation accumulation lines means over time, suggesting weaker stabilizing selection. However, if direct stabilizing selection is not acting on bristle number, there is no reason to expect concordance of estimates from the two experiments. Causal relationship of bristle number to fitness: Is

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Nuzhdin et al.

the observed intermediate optimum forbristle number due to stabilizing selection acting directly on bristle number phenotype, apparent stabilizing selection caused by deleterious pleiotropic fitness effects of mutations affecting bristle number, or overdominance of mutant alleles affecting bristle number? Classical overdominance for fitness at loci affecting bristle number can be ruled out as a potential cause of apparent stabilizing selection, because this mechanism would not be expected to cause an intermediate optimum for the pure lines, unless all of the unselected lines had accumulated mutations at bristle lociwith overdominant fitness effects. This is unlikely, and would not cause the declining covariances of line meansover time observed for the unselected lines ( MACKAY et al. 1995) . Furthermore, overdominance would result in heterozygous genotypes exhibitinganintermediateoptimumfor fitness. Whilethis is true for sternopleuralbristle number, it is not for abdominal bristle number, where there is a linear relationship between bristle number andfitness with the most fit genotypes from crosses among high selected lines. It remainsto distinguish between direct selection acting on deviation of bristle number phenotype from the optimum, and deleterious pleiotropic side effects on fitness of mutations affecting bristle number as the cause of the observed intermediateoptimuminthe pure lines, by examining bristle numbers and fitnesses of progeny produced from all possible crosses of selection lines. Recall that if direct stabilizing selection acts on bristle number, then ( 1) there will be an intermediate optimum for fitness in both pure lines and crosses and ( 2 ) the fitness values will be the same for pure lines and crossed lines with the same bristle score. The latter point depends on the degree of dominance of fitness effects ofslightly deleterious mutations unrelated to bristle number that have accumulated in the selection lines. If they are strictly recessivethen complementing backgroundfitness effects could confound the interpretation of direct stabilizing selection. However, viability effects of slightly deleterious spontaneous mutations are practically additive ( CROW and SIMMONS 1983), and themean fitness of unselected lines (0.46) is not significantly different from the mean fitness of crosses among unselected lines (0.50; ko = 0.33, P > 0.05) in our experiment, suggesting the effects of complementing background mutationswill not appreciably confound the interpretationof selection on loci affecting bristle number. For neither bristle trait are both conditions for direct stabilizing selection met. For sternopleural bristles there was a significant quadratic component to the regression of squared deviation from the optimum on fitness of progeny from crosses among selection lines, indicating heterozygous individuals with intermediate bristle number phenotypes were more fit than phenotypic extremes, as for the purelines. However, the aver-

age fitness of the H X H and L x L cross progeny was twice as high as the average fitness of the pure lines, which is not consistent with direct stabilizing selection only operating on sternopleural bristle number. For abdominal bristle number, the patternof an intermediate optimum for fitness observed for the pure lines is not observed for theprogeny of crosses among selection lines, so direct stabilizing selection cannot account for both sets of observations. A case could be made for direct selection against low abdominal bristle number, because the average fitness of the L x L line crosses is equivalent to that of the purelines, but notagainst high abdominal bristle number, because theH X Hline cross progeny are twice as fit as the pure high lines. If apparent stabilizing selection is caused by the pleiotropic connection between mutant effects on bristle number and fitness, the average fitness of all possible crosses of H X H, H X L and L X L selection lines should be equal, independently of p, the correlation between the magnitude of bristle effects and decline of fitness. This simple prediction does, however, assume that the degree of dominance for fitness does not vary with effect on the bristle trait. The observed relationship between sternopleural bristle number and fitness in both pure lines and crosses among selected lines is thus consistent with the pleiotropic prediction, with partly recessive fitness effects of mutations affecting both high and low sternopleural bristle number. There is an overall association between the deviation of sternopleural bristle number and fitness in the progeny of crosses among selection lines, due entirely to progeny of reciprocal crosses of high selection replicates 2 and 3, so one cannot rule out some component of direct selection acting on this trait. The observed linear relationship between abdominal bristle number and fitness in progeny of crosses does not fit the simple prediction of the pleiotropic model. However, fitness effects of mutations affecting abdominal bristle number are variable: fitness effects of mutations that increase abdominal bristle numberare recessive, whereas those that reduce abdominal bristle number are additive. Given these variable degrees of dominance for fitness, a linear relationship between abdominal bristle numberand fitness in progeny of crosses iswhat would be expected under the pleiotropic model. Caveats: The above interpretation is based on two assumptions; ( 1 ) that the selection lines have accumulated mutations at different loci affecting bristle number and ( 2 ) that the selected and unselected lines have the same background level of slightly deleterious mutations unrelated to bristle number. To determine the degree to which replicate selection lines had accumulated mutations affecting bristle number at different loci, reciprocal crosses were made at generation 94 between all pairs of lines selected in the same direction for the same trait, and directional selection was continued for nine further generations from the F2 of each

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Selection Mutations on Polygenic

cross. In general, the final responses of the synthetic lines did not exceed the mean bristle number of the most extreme parental line( FRYet al. 1995) .This observation is consistent with either allelism of, or epistatic interactions between, bristle effects of mutationsin lines selected in the same direction for the same trait. Violation of the assumption that mutations affecting bristle loci are atdifferent loci in the mutation accumulation lines does not affect our interpretation that there is an intermediate optimum for bristle number in the pure lines. Nor does it affect the interpretation that neither overdominance nor direct stabilizing selection explain the relationships between bristle numbers and fitness in both pure lines and in progeny of crosses among selection lines. However, violating this assumption could affect the details of the pleiotropic interpretation of apparent stabilizing selection. If fitness effects of mutations atthe same loci or atdifferent epistatically interacting loci in two lines also fail to complement, or interact epistatically, then thefitness of crossesbetween these lines would be equal to that of the pure lines, giving the appearance of direct selection, additivity of fitness effects or a pleiotropic correlation between the magnitude of the deviation from the optimum bristle number and the extent of the decline in fitness. Violation of assumption ( 1 ) may account for thelow fitness of progeny from reciprocal crosses of high sternopleural replicates 2 and 3, and of all crosses of low abdominal selection replicates. High sternopleural selection replicates 2 and 3 have both accumulated mutations at theemc bristle locus ( LINDsLEY and ZIMM 1992) , as the progeny of crosses of these lines to a stock containinga deficiency of emc have the emc phenotype (T. F. C. MACKAY, unpublished data). Thus thelow fitness of progeny from reciprocal crosses of these lines might be attributable to mutations at the emc locus. Reciprocal crosses of these lines account for thesignificant quadraticregression of deviation of bristle number from the optimum on fitness of progeny of crosses of sternopleural bristle selection lines. Therefore, if the sternopleural bristle selection lines have other alleles in common or thatinteract,their fitness effects are not obviously associated with bristle effects. Progeny of crosses among lines selected for reduced numbers of abdominal bristles had on average the same low fitness as the pure selection lines. We cannot exclude the possibility that these lines have mutations at the same loci with noncomplementing fitness effects, but in this case the locihave not been identified. If the low abdominal bristle selection lines have accumulated mutations at common bristle loci, then our interpretation of the linear relationship between bristle number and fitness in the progeny of crosses among selection lines as being due to variable degrees of dominance of fitness effects of mutations affecting bristle number may not be correct. The initially isogenic selected and unselected muta-

tion accumulation lines were assumed to have similar background levels of slightly deleterious mutations unrelated to bristle number because they were maintained atthe same census size for approximately the same number of generations. However, it is possible that hitchhiking of deleterious mutations closely linked to selected mutations affecting bristle number could lead to a higher loadof background mutations in the selection lines and contribute to their reduced fitness. It is not possible to estimate the extentto whichhitchhiking has confounded the interpretation of an intermediate optimum for bristle number based on the comparison of fitness of pure selection lines and unselected lines. The qualitative consistency of the estimates of the strength of selection from the experiments reported here and from analysis of the evolution of variances and covariances of unselected line means, to which the hitchhiking explanation does not apply ( MACKAY et al. 1995), gives some confidencethat our inference of selection against mutations with extreme effects on bristle number in selection lines is correct. Pleiotropic models: There is strong empirical evidence that new P-element insertional mutations ( MACKAYet al. 1992b) and spontaneous mutations ( CABALLERO et al. 1991; SANTIAGO et al. 1992; LOPEZand LOPEZ-FANJUL 1993) have deleterious pleiotropic effects on theviability component of fitness. Simple models incorporating pleiotropic side-effects on fitness of mutations affecting quantitative traits to predict equilibrium genetic variance maintained by a balance between selection and mutation also predict apparentstabilizing selection, but of too small a magnitude to account for the strongstabilizing selection observed in natural populations ( BARTON1990; KEIGHTLEY and HILL1990; CABALLERO and KEICHTLEY 1995) . Strong apparent stabilizing selection will only occur with pleiotropic models of mutation-selection balance if thecorrelation between absolute mutant effects on the trait and fitness are very high ( KEIGHTLEY and HILL1990; CABALLERO and KEICHTLEY 1995), orif there is synergistic epistasis for fitness effects of new mutations ( KONDRASHOVand TURELLI 1992; GAVRILETS and DE JONG 1993). Because we have inferred apparent stabilizing selection on new mutations affecting bristle number of comparable strength to that commonly quoted for natural populations, it is likely that the pleiotropic correlation of mutant effects on bristles and on fitness is high and/or there is synergistic epistasis for fitness in the selection lines. Further experiments using isogenic lines with single mutations affecting bristle number should elucidate the relative importance of these factors in producing apparent stabilizing selection. This workwas supported by National Institutes of Health grants GM45344 and GM-45146 to T.F.C.M. and from the Frontiers in Genetics Program of the Russian Academy of Sciences to S.V.N.

LITERATURE CITED BARTON,N. H., 1990 Pleiotropicmodels of quantitative variation. Genetics 124: 773-782.

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A., and P. D. KEIGHTLEY, 1995 A pleiotropic model of mutation inDrosophila melanogmter: Nonlinear divergence among variation in quantitative traits. Genetics 138: 883-900. unselected strains. Genetics 139: 000-000. CABALLERO, A., M. A. TORO and C. L;AAOPEZ-FANJUL, 1991 The MATHER, K., 1941 Variation and selection of polygenic characters. response to artificial selection from new mutations in Drosophila J. Genet. 41: 159-193. melanogaster. Genetics 127: 89-102. NUZHDIN,S. V., and T. F. C. MACKAY, 1994 Direct determination CLAWON,G. A,,J. A. MORRISand A. ROBERTSON, 1957 An experimenof retrotransposon transposition rates in Drosophila melanogaster. tal check on quantitativegenetical theory. I. Short-term reGenet. Res. 63: 139-144. sponses to selection. J. Genet. 55: 131-151. ROBERTSON, A,, 1955Selection in animals: synthesis. Cold Spring CROW, J. F., and M. J. SIMMONS, 1983 The mutation load Drosophila, in Harbor Symp. Quant. Biol. 2 0 225-229. pp. 1-35 in The Genetics and Biology of Drosophila, edited by H. L. ROBERTSON, A,, 1967 The nature of quantitative variation, pp. 265Carson, M. Ashburner and J. N. Thompson. Academic Press, 280 in Hetitagefrom Mendel, edited by A. BRINK.The University London. of Wisconsin Press, Madison, WI. ROBERTSON, A,, 1970 The state of quantitative genetics in relation FRY,J. D., K. DE RONDEand T.F. C. MACKAY, 1995 Polygenic mutation to the real world, pp. 2-17 in Proceedings of the 19th National in Drosophila mlanogaster: genetic analysis of selection lines. GeBreeders Roundtable, Kansas City, MO. netics 139(3) (in press). ROFF, D. A,, and T. A. Moussmu, 1987 Quantitative genetics and S., and G. DEJONG,1993 Pleiotropic models of polygenic GAVRILETS, fitness: Lessons from Drosophila. Heredity 58: 103-118. variation, stabilizing selection and epistasis. Genetics 134: 609A. DOMINGUEZ, M. A. TOROand C. LOPEZSANTIAGO, E.,J. ALBORNOZ, 625. FANJUL,1992 The distribution of effects of spontaneous mutaHARTL,D. L., and H. JUNGEN,1979 Estimation of average fitness of tions on quantitative traits and fitness in Drosophila melanogaster. populations ofDrosophila melanogaster and theevolution of fitness Genetics 132 771-781. in experimental populations. Evolution 3 3 371-380. SAS INSTITUTE, INC.,1988 SAS/STAT User’s Guide,Release 6.03Edition. HAYMER, D. S., and D. L. HARTL, 1983The experimental assessment SAS Institute Inc., Cary, NC. of fitness in Drosophila. 11. A comparison of competitive and nonJ. G. C., 1974 The effects of larval competition on a quantitaSPIERS, competitive measures. Genetics 104 343-352. tive character in Drosophila mlanogaster. Ph.D. Thesis, University HILL,W. G., and P. D. KEIGHTLEY, 1988 Interrelations of mutation, of Edinburgh, Edinburgh. population size, artificial selection and naturalselection, pp. 57SVED,J. A,, 1971 An estimate of heterosis in Drosophila melanogaster. 70 in Proceedings of the 2nd International Conference on Quantatative Genet. Res. 18: 97-105. and Genetics, edited by B. S. WEIR, E.J. EISEN,M. M. GOODMAN SVED,J. A., 1975 Fitness of third chromosome homozygotes in DroG. NAMKOONG. Sinauer, Sunderland, MA. sophila melanogaster. Genet. Res. 25: 197-200. KEARSEY, M.J., and B. W. BARNES, 1970 Variation for metrical characTURELLI,M., 1984 Heritable genetic variation via mutation-selection ters in Drosophila populations. 11. Natural selection. Heredity 25: balance: Lerch’s ~ c t meets a the abdominal bristle. Theor. Popul. 1 1 -21. Biol. 25: 138-19:;. KEIGHTLEY, P. D., and W. G. HILL, 1990 Variation maintained in quantitative traits with mutation-selection balance: pleiotropic Communicating editor: M. LWCH side-effects o n fitness traits. Proc. R. SOC.Lond. Ser. B 242: 95100. KNIGHT,G. R., and A. ROBERTSON, 1957 Fitness as ameasurable character in Drosophila. Genetics 42: 524-530. APPENDIX A KONDRASHOV, A. S., and M. TURELLI,1992 Deleterious mutations, apparent stabilizing selection and the maintenance of quantitaCompetitive indices and bristle numbers tive variation. Genetics 132: 603-618. of unselected mutation accumulation lines LATTER, B. D. H., andA. ROBERTSON, 1962 The effects of inbreeding and artificial selection on reproductive fitness. Genet. Res. 3 Line N CI ST AB 110-138. LINDSLEY, D.L., and G.G. ZIMM,1992 The Genome of Drosophila 1 818 0.440 +- 0.047 16.1 2 0.22 15.4 +- 0.31 melanogaster. Academic Press, San Diego. 2 716 0.398 2 0.056 15.5 t 0.18 15.4 2 0.25 LINNEY,R.,B.W. BARNESand M. J. W E Y , 1971 Variation for 0.280 ? 0.092 14.8 +- 0.13 15.6 2 0.26 543 3 metrical characters in Drosophila populations. 111. The nature of 15.0 t 0.15 15.3 2 0.28 1062 4 0.295 2 0.052 selection. Heredity 27: 163-174. 5 781 0.660 2 0.039 16.1 t 0.25 15.1 2 0.20 LOPEZ,M. A., and C. LOPEZ-FANJUI., 1993 Spontaneous mutation for 0.241 2 0.063 15.8 2 0.16 16.1 2 0.39 6 a quantitative trait in Drosophila mlanogaster. 11. Distribution of614 mutant effects on the trait and fitness. Genet. Res. 61: 117-126. 888 15.0 2 0.28 7 0.611 2 0.108 15.2 t 0.14 MACKAY, T. F. C., R. F. LYMAN,M. S. JACKSON, C. TERZIAN and W. G. 844 0.654 2 0.085 15.2 2 0.15 15.8 2 0.25 8 HILL,1992a Polygenic mutation in Drosophila melanogaster: esti9 771 0.575 2 0.062 15.0 t 0.15 15.0 +- 0.26 mates from divergence among inbredstrains. Evolution 46: 3000.444 +- 0.085 16.0 2 0.18 14.3 2 0.28 667 10 316. MACKAY, T. F. C., R.F. L m and M. S. JACKSON,1992b Effects of Values are means 2 SE. N is the total number of flies on Pelement insertions o n quantitative traits in Drosophila melanogaswhich the competitive indices (CI) are based, with SEs comter. Genetics 130: 315-332. puted from the variance among replicate vials. Sternopleural MACKAY, T. F. C., J. D.FRY, R. F.LYMAN and S. V. NUZHDIN,1994 (ST) and abdominal (AB) bristle numbers are averaged over Polygenic mutation in Drosophila melanogaster: estimates from re20 individuals of each sex and SEs are based on variance sponse to selection of inbred strains. Genetics 136 937-951. among individuals. MACKAY, T. F. C., R.F. LYMAN and W. G. HILL, 1995 Polygenic CABALLERO,

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APPENDIX B Competitive indices and bristle numbersof progeny of crosses among unselected mutation accumulation lines Line Cross

(9

x d)

1x2 2 1 3 1 4 2 3 2 4 4 3

x1 x 3 x 1 x 4 x 1 x 3 x 2 x 4 x 2 x 3 x 4

N

CI

911 645 1307 979 1169 439 505 476 1058 663 473 1137

0.459 rt 0.066 0.519 t 0.050 0.631 t 0.039 0.420 t 0.068 0.494 t 0.042 0.460 t 0.075 0.459 t 0.071 0.393 t 0.066 0.510 t 0.047 0.542 t 0.067 0.571 t 0.055 0.529 2 0.049

AB

ST 15.8 2 15.6 C 16.1 C 15.0 +15.4 2 16.0 +15.3 2 15.2 2 15.6 2 15.9 +15.4 2 15.2 +-

0.24 0.16 0.19 0.18 0.17 0.16 0.15 0.17 0.17 0.16 0.18 0.14

15.3 t 0.28 14.6 t 0.32 14.9 t 0.26 14.7 t 0.25 14.3 t 0.24 14.2 t 0.26 14.9 t 0.29 15.8 rt 0.29 15.6 t 0.33 14.9 rt 0.29 14.8 t 0.29 15.7 rt 0.30

Values are means t SE. Line numbers correspond to lines 1-4 of APPENDIX A. Other statistics are also as for APPENDIX A.

APPENDIX C Competitive indices and bristle numbers of mutation accumulation lines selected for sternopleural bristle number and crossesamong them

d H1 N CI ST H2 N CI ST H3 N CI ST L1 N CI ST L2 N CI ST L3 N CI ST

H1

H2

H3

L1

12

13

553 0.287 t 0.071 17.8 rt 0.22

746 0.405 t 0.085 21.5 t 0.45

1058 0.474 t 0.053 19.4 t 0.27

544 0.479 t 0.060 16.0 t 0.17

756 0.427 rt 0.067 15.2 t 0.19

1356 0.521 t 0.027 15.9 t 0.19

754 0.579 t 0.063 21.3 t 0.34

547 0.161 t 0.068 24.3 t 0.44

842 0.139 t 0.063 23.2 t 0.58

569 0.471 t 0.084 17.9 rt 0.27

1093 0.325 t 0.095 16.4 t 0.23

902 0.383 rt 0.082 18.1 t 0.35

1072 0.354 t 0.076 19.3 t 0.28

685 0.151 t 0.042 25.0 t 0.54

748 0.153 t 0.055 23.9 t 0.40

1282 0.481 t 0.068 17.0 t 0.19

683 0.289 t 0.050 16.2 t 0.23

1173 0.343 0.033 16.5 t 0.23

681 0.387 t 0.070 16.3 2 0.22

600 0.454 2 0.044 18.8 t 0.35

960 0.507 t 0.050 17.3 t 0.22

539 0.276 t 0.053 14.0 t 0.18

552 0.376 t 0.092 13.9 t 0.21

1160 0.377 t 0.060 14.0 rt 0.14

649 0.393 t 0.073 15.8 t 0.17

807 0.445 rt 0.057 17.2 t 0.25

998 0.477 t 0.049 16.9 t 0.16

865 0.393 t 0.054 14.1 t 0.16

458 0.185 t 0.031 10.8 t 0.12

1070 0.415 t 0.044 13.7 t 0.14

945 0.531 t 0.081 16.2 2 18.7 0.17

623 0.369 t 0.075 _t 18.0 0.27

1059 0.400 t 0.052 ? 13.8 0.21

746 0.470 t 0.077 t 0.15

1134 0.320 t 0.066 13.4 2 12.7 0.16

656 0.066 t 0.042 t 0.15

Values are means 2 SE. H1-H3 and Ll-L3 are independent replicate lines selected for high and low sternopleural bristle number, respectively. Other statistics are as for APPENDIX A.

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Nuzhdin et al.

APPENDIX D Competitive indices and bristle numbersof mutation accumulation linesselected for abdominal bristle number and crosses among them 0 6 H1 N CI

AB H2 N CI

AB

H1

H2

H3

L1

12

13

506 0.153 t 0.047 19.0 rt 0.35

963 0.693 2 0.038 18.8 t 0.25

799 0.575 2 0.058 18.5 ? 0.27

664 0.204 t 0.050 13.5 2 0.35

561 0.247 2 0.054 12.2 rt 0.37

546 0.315 2 0.055 15.4 2 0.54

655 0.337 t 0.067 19.4 ? 0.32

602 0.349 2 0.072 18.1 ? 0.24

502 0.585 ? 0.085 17.9 ? 0.29

509 0.369 t 0.066 13.3 2 0.37

469 0.216 2 0.031 11.8 f 0.12

399 0.383 t 0.043 16.1 2 0.50

868 0.318 t 0.075 18.3 t 0.29

974 0.526 ? 0.068 17.8 ? 0.20

708 0.203 2 0.035 17.6 5 0.28

497 0.223 2 0.070 12.9 ? 0.39

447 0.293 5 0.062 10.3 2 0.25

729 0.460 rt 0.090 15.2 t 0.53

555 0.298 t 0.071 11.4 rt 0.43

765 0.398 rt 0.058 12.9 ? 0.39

566 0.279 t 0.060 11.8 t 0.50

816 0.006 t 0.004 2.1 ? 0.22

679 0.125 2 0.042 4.6 2 0.37

593 0.241 2 0.069 9.5 ? 0.44

616 0.181 t 0.044 8.4 t 0.35

1043 0.302 t 0.078 11.7 ? 0.36

545 0.282 2 0.065 10.3 ? 0.25

445 0.190 t 0.049 4.3 2 0.32

492 0.051 t 0.027 4.8 2 0.31

547 0.161 2 0.050 6.5 2 0.43

786 0.431 t 0.077 14.7 5 0.45

1063 0.568 t 0.063 13.9 2 0.60

611 0.461 ? 0.075 15.4 2 0.32

598 0.230 t 0.049 8.3 2 0.37

714 0.120 t 0.046 6.8 2 0.39

538 0.263 t 0.050 8.4 ? 0.58

H3

N CI

AB L1 N CI

AB L2 N CI

AB L3 N CI

AB

Values are means t SE. Hl-H3 and Ll-L3 are independent replicate lines selected for high and low abdominal bristle number, respectively. Other statistics are as for APPENDIX A.