SEGREGATIONAL LOAD IN DROSOPHILA KIKJSAWAI. 111. NATURAL POPULATIONS1
NEWTON FREIRE-MAIA AND ADEMAR FREIRE-MAIA2 Laboratdrio de Gendtica Humana, Faculdade de Filosofia, Universidade do Parana, Curitiba, Pr, Brasil
Received April 24, 1964
ATURAL populations of D.kikkawai Burla (previously cited as D.montium NdeMeijere) present three different types of polymorphism: (1) a polymor- phism (associated with a sexual dimorphism) involving color and pattern of abdominal tergites; (2) a chromosomal polymorphism expressed in a long para- centric inversion in the left arm of chromosome 11; (3) a chromosome hetero- morphism in consequence of the mitotic chromosome IV being found both as metacentric and as acrocentric. With respect to color and pattern of abdominal tergites, two genetically pure forms (light and dark) have been isolated. Crossing experiments showed that a single pair of autosomal alleles is mainly responsible for the differences between them, with dark pigmentation dominant. The two color forms are easily distin- guished among females, but with difficulty among males. (For data, see FREIRE- MAIA1964a,b.) In this paper, data on the color polymorphism and on the inversion polymor- phism in natural populations will be presented and discussed. The problem of the chromosome heteromorphism will be only briefly touched, since no quantita- tive information was obtained concerning it.
MATERIALS AND METHODS
Domestic populations of Drosophila have betn survcyzd fr3m Buenos Aires. Argentina, to northeastern and nsrthern Brazilian localities. In a number of these places, D.kikkauai either was not found or was present with extremely low frequency. The localities where this species has been found are shown in Figure 1. The populations were sampled during about 11 years (1947-1957.). The majority of the collections have been made, however, during the period 1951-1957. Collections of the flies were made in markets, orchards, fruit deposits, etc. using an entomological net with a narrow end. No artificial “bait” has been used. The flies have been analysed in three different ways, using: (1) collected females; (2) cross- ings of collected males to light females from laboratory strains; and (3) the offspring of females inseminated in nature. For the analysis of chromosomes, preparations by the usual orcein method have been used. The vials with flies were kept at about 25°C. Quantitative data were obtained
Dedicated to Dn. HARRYM. MILLER,JR., ex-director of the Rockefeller Foundation, in recognition of his outstanding contribution to the development of genetics in Brazil. This research has been made under grants from the Rockefeller Foundation, the National Research Council of Brazil, the National Nuclear Energy Commission of Brazil, and the Re- search Council of the University of Parani. ‘ Present address. Faculdade de CiBncias MQdicas e Biol6gicas, Botucatu, SP, Brasil.
Genetics 50: 789-802 h-ovember 1964 790 N. FREIRE-MAIA AND A. FREIRE-MAIA
FIGURE1.-Map of Brazil showing the localities where collections have been made. 1. Porto Alegre; 2. Florian6polis; 3. Ascurra; 4. Blumenau; 5. Gaspar; 6. Itajai; 7. Joinville; 8. Para- nag&; 9. Morretes; 10. Antonina; 11. Curitiba; 12. Irati; 13. Londrina; 14. Santos; 15. s. Paulo; 16. Campinas; 17. Assis; 18. Presidente Prudente; 19. Lins; 20. Belo Horizonte; 21. GoiCnia; 22. Salvador. through the analysis of only one larva from each female captured in nature. Nonfertilized or sterile females were discarded.
RESULTS Color polymorphism-The data. Method I: Table 1 presents the results of a number of samples collected in different localities as analysed by Method 1 (direct examination of females) during the period 1947-1957. Statistical analysis of the comparisons among the different collections within the same locality revealed that the only one showing a significant difference (with P close to the 0.05 level) is ParanaguA. The only collection departing from the mean frequency 0.57 is that made in September 1952, with 0.67 light females (x2= 6.34; P between 0.01 and 0.02). The value of x2 for the 16 groups of data shown in Table 1 is 167.77 (d.f. = 15; P I,11cahtv Time n* Light females Light females (Total)+ Porto Alegre, RGS Febr. '55 61 0.74 F 0.06 0.76 t 0.05 Florianbpolis. SC July '54 27 0.63 * 0.09 SeDt. '55 41 0.54 f 0.08 0.58 f 0.06 Itajai, SC Juiy '54 63 0.62 f 0.06 Febr. '56 62 0.71 i 0.06 Jan.-March '57 27 0.74 i- 0.08 0.67 i 0.04 Gaspar. SC June '52 14 0.43 i 0.13 Jan.-March '57 12 0.42 F 0.14 0.56 f 0.08 Ascurra, SC July '54 12 0.92 t 0.08 Oct. '55 16 0.69 t 0.12 March '57 4.5 0.53 +_ 0.07 0.64 k 0.05 Blumenau. SC Febr. '56 24 0.67 F 0.10 Dec. '56 23 0.65 t 0.10 March '57 26 0.69 f 0.09 0.69 t 0.05 Paranagui, Pr March '52 13 0.62 t 0.13 Sept. '52 148 0.67 F 0.04 Oct. '53 14 0.43 t 0.13 Sept. '54 25 0.60 * 0.10 Oct. '54 139 0.50 k 0.04 Nov. '55 24 0.58 F 0.10 Dec. '56 111 0.50 -C 0.05 0.57 F 0.02 Morretes. Pr March '47 33 0.94 F 0.04 Apr.-July '51 16 0.94 i 0.06 Sept.-Oct. '51 37 0.81 i 0.06 Nov.-Dec. '51 42 0.86 F 0.05 Aug. '53 22 0.77 F 0.09 Oct. '53 17 0.88 t 0.08 April '54 72 0.89 i. 0.04 Sept. '54 47 0.79 t 0.06 Oct. '54 191 0.90 t 0.02 Nov.'55 21 1.oo 0.88 t 0.01 Antonina, Pr Nov. '51 26 0.92 t 0.05 0.91 t 0.05 Londrina, Pr Sept. '54 16 0.88 F 0.08 0.85 t 0.08 S. Paulo, SP June '47 168 0.76 t 0.03 March '49 73 0.86 i 0.04 June-July '49 46 0.87 f 0.05 May '54 20 0.75 i 0.10 0.80 F 0.02 Presidente Prudente. SP July '56 13 0.69 F 0.13 0.69 i 0.13 Lins, SP April-May '54 46 0.89 k 0.05 0.89 i 0.05 Belo Horizonte, MG Aug.-Sept. '53 26 0.81 & 0.08 0.81 t 0.08 Goibnia, Go Sept. '53 25 0.84 k 0.07 0.84 * 0.07 Others$ - 10 0.90 t 0.09 0.90 f 0.09 * n is the t,,tal n~m111erof females. Only the samples with n > 10 are given. -IUnder tlir heading "Light females (Total),'' the frequencies of light females in the total of the siiniplt~collected in each localit)-. including those with n < 10. are given. $ "Others' lnclnde Curitiba (Pr). Irati (Pr), Santos (SP), Assis (SP), and Salvador (Ba). Collections in these locali- ties h.i\r hwn made in July 1951. April 1952, December 1954. September 1954. and Februarv 1954. respectively. RGS-Rco Gianrle do SUI; SC-Santa Catarina; Pr-ParanB; SP- Sdo Paulo; RIG-&has Gerais; Go-GoiBs. localities (from Porto Alegre to ParanaguA) than in the other ones (from Mor- retes in the South to Salvador in the East and Goidnia in the Center-West) (ex- cepting the small sample of Presidente Prudente). When comparison is made among the seven southmost localities (here called group A) and among the other ones (group B), the values of xz are respectively 16.91 (d.f. = 6; P+O.Ol) and 14.96 (d.f. = 8; P>0.05). The data from Porto Alegre contribute with a x2 equal to 6.55 to the value of 16.91. A reanalysis of group A without the data from Porto Alegre led to a x2 equal to 9.70 (d.f. = 5; P>0.05). It may be said therefore that 792 N. FREIRE-MAIA AND A. FREIRE-MAIA the Porto Alegre data are an exception within group A, characterized by a mean frequency of light females around 0.60. The whole group B, with a mean fre- quency of light females around 0.85, could be accepted as a relatively homogenous one. The total frequency of light females in S. Paulo (0.80) may be considered, however, as a law one on the basis of its own individual value of x2 (6.73; P TABLE 2 Frequencies of light females in diflerent months Months A(1)' B(1) A@) B(3) A(1) B(l) A(2) B(3) January 0.50 0.50 February 0.71 0.67 0.70 t ] 0.67 0.89 0.65 0.95 March 0.64 0.90 0.62 0.95 J April 0.86 0.90 0.86 0.90 May 0.79 0 85 ] 0.50 0.82 0.50 0.89 June 0.37 0.79 0.37 0.90 1 July 0.67 0.76 0.67 0.76 August 0.79 0.79 ] 0.65 0.81 0.65 0.81 September 0.64 0.82 0.64 0.82 October 0.55 0.89 0.55 0.89 November 0.63 0.92 0.60 0.92 1 0.55 0.89 0.54 0.89 December 0.53 0.79 0.53 0.79 J X' X' 23.66 21.92 19.05 13.91 13.89 10.76 10.43 11 07 d.f. 9 10 9 10 3 3 3 3 P * A and B refer to the southmost and to the other populations respectirely; (1) All the data; (2) Excluding Porto Alegre; (3) Excluding SHo Panlo. $ Only one light female. POLYMORPHISM IN D. kikkawai 793 TABLE 3 Distribution of the three genotypes among males in several Brazilian localities (expected numbers according io the Hardy-Weinberg formula) Frequency Gene Localitv cc CC cc Total of cc frequency (CI X' Porto Alegre, RGS 3 10 33 46 0.72 .83 2.72 Expected 1.39 13.22 31.39 46 Florian6polis, SC 2 38 47 87 0.54 .76 3.24 Expected 5.05 31.83 50.12 87 Itajai, SC 4 40 83 127 0.65 .81 0.10 Expected 4.54 38.93 83.53 127 Ascurra, SC 3 21 56 80 0.70 .83 0.33 Expected 2.28 22.47 55.25 80 Gaspar, SC 0 9 14 23 0.61 .80 1.36 Expected 0.88 7.25 14.87 23 Blumenau, SC 4 12 37 53 0.70 .81 3.59 Expected 1.89 16.25 34.86 53 Paranagui, Pr 29 137 135 301 0.45 .68 0.46 Expected 31.60 131.85 137.55 301 Morretes, Pr 1 46 335 382 0.88 .94 0.19 Expected 1.52 45.10 335.38 382 SQo Paulo, SP 0 6 23 29 0.79 .90 0.39 Expected 0.31 5.36 23.33 29 Lins. SP 0 3 36 39 0.92 .96 0.06 Expected 0.06 2.85 36.09 39 Others* 1 9 31 41 0.76 .87 0.12 Expected 0.74 9.52 30.74 41 Total 47 331 830 1208 0.69 .82 3.64 Expected 37.38 350.24 820.38 1208 * .JoinwIk. SC 10. 1. 0 : .Intonma, Pr (0, 2. 11); Londrina, Pi- (0, 3, Y); Santos. SP (1, 3, 2); Campinas, SP (0. 0, I ' : .\>>IS, SI' (0. 0. 5 '. and Presidente Prudente, SP (0; 0, G), expected, the same fact that emerged in the analysis of the samples of light females (Table 1) is clearly seen in Table 3, i.e., that allele c is commoner in the localities of group B than in those of group A. For the distribution of gene frequencies in 794 N. FREIRE-MAIA AND A. FREIRE-MAIA the southmost localities (group A) x2 = 34.07 (d.f. = 6; P TABLE 4 Frequencies of the light genotype (male plus females) in differentpopulations Locality N Light genotype Porto Alegre 124 0.74 1 Florian6polis 163 0.56 I Itajai 295 0.66 Gaspar 59 :::: I Ascurra 158 0.67 I Blumenau 131 0.69 J Morretes 886 SBo Paulo 343 0.80o.88 1 Lins 85 O”’ 0.86 Antonina 45 0.89 Lonririna 32 Presidente Prudente 19 0.810.79 i POLYMORPHISM IN D.kikkawai 795 males crossed to light females from laboratory strains. A total of 7,627 flies has been analysed. This number includes the offspring of males from Paranagu6, Blumenau, Gaspar and Itajai. The sex ratio (s.r.) has been found to be about the same (around 0.48) in the three kinds of progenies produced, namely those com- posed of (a) light and dark flies (s.r. = 0.49; N = 2,823), (b) light flies only (s.r. = 0.48; N = 4,173) and (c) dark flies only (s.r. = 0.47; N = 631). x2 for the comparison of the three groups equals 0.39 (d.f. = 2; P>0.80). Method 3: According to this method, the phenotype of the offspring of females inseminated in nature was determined in order to obtain some information on the genotype of the males with which they have copulated. Under the assump- tion that each collected light female has been inseminated only once, this method turns out to be a replica, made in nature, of our Method 2. The data are shown in Table 5. With the exception of the data from Paranaguti and Florian6polis (having significant deviations at the 0.05 level), all the others and the “grand total” led to results which are comparable to those obtained with Method 2. With the exception only of Paranaguti, all the others show distributions in accordance with the Hardy-Weinberg Law (Table 5). The analysis of the offspring of dark females would not give the same kind of information shown in Table 5, since it is impossible to discriminate pheno- typically between females which are homozygotes (CC) and heterozygotes (Cc), and therefore impossible to discriminate between the different types of crosses. In spite of this unfavorable situation, the offspring of dark females inseminated in natural populations has also been analysed. The data show that the frequen- cies of heterozygous females in the total of dark females are either equal to or higher than 0.70 in all the samples. Chromosomal polymorphism-The data: In Japanese as well as in Brazilian populations, previous investigations ( OSIMA 1940; FREIRE-MAIA1947) failed to detect any inversions. Later, we had the opportunity of examining the salivary TABLE 5 Distribution of the three genotypes among males as determined through the analysis of the offspring of light females inseminated in natural populations Numbers Frequencies CC Cc cc Total cc C Florianopolis, SC 3 11 8 22 0.36 0.61 6.16 <0.05 0.06 0.80 Itajai. SC 1 20 42 63 0.67 0.83 0.41 >0.80 1.07 0.30 Ascurra, SC 4 18 22 44 0.50 0.70 5.19 >0.05 0.01 >0.90 Blumenau, SC 1 12 23 35 0.64 0.81 1.88 >0.30 0.15 0.70 Paranaguj. Pr 1 40 32 73 0.44 0.71 6.08 <0.05 8.29 <0.01 Morretes. Pr 0 20 103 123 0.84 0.92 1.76 >0.30 0.96 >0.30 Grand Total* 10 131 288 429 0.67 0.82 3.43 >O.lO 1.20 >o.20 * “Grand Total” also includes data from Porto hlegre, (0, 2, 15), Gaspar (0, 2, 6), Antonina (0, 1, l), Londrina 1.0. 3. 81. Lins (0,. 0, 9),.S.,Paulo(0, 0, 11). Presidente Prudente (0, 1, 8) and Assis (0, 1, 0). Under Frcquencles, cc indicates the frequencies of light females: c indlcates the frequencies of the allele r for light I’:gmentstion: (1) refers to the comparison between the data in this table and those frmn Table 3; (2) refers to the comparison between the data presented here and the expected values according to the Hardy-W’einbel-g Law. 796 N. FREIRE-MAIA AND A. FREIRE-MAIA -/.\ FIGURE2.-Paracentric inversion in the left arm of chromosome 11. gland chromosomes of a sample from Honolulu, Hawaii, and of the offspring from this sample crossed to Brazilian flies; no chromosomal rearrangement has been observed. In a stock from S. Paulo, SP, however, we later found a long median inversion in the left arm of chromosome I1 (cf. map in FREIRE-MAIA l947), comprising approximately half the arm (Figure 2). New attempts to find this inversion in the same stock and in flies collected before 1954, in the same city, have been unsuccessful. From 1951 up to 1955, a survey on the incidence of chromosomal variation in domestic species of Drosophila led us to determine the frequency of that inversion in natural populations of D.kikkawai from different Brazilian regions. The find- ings in all the species which mostly compose the domestic fauna may be seen in FREIRE-MAIA(1955a,b, 1961) and FREIRE-MAIA,ZANARDINI and FREIRE-MAIA (1953). Detailed information on these findings in D. kikkawlai is presented in Table 6. Variations within the same locality are nonsignificant. Comparison among the totals of the 11 groups shown in Table 6 gives x2 = 20.56 (d.f. = IO; P<0.05). There is, however, only one sample (Itajai) departing significantly from the general distribution (x2= 7.34; P TABLE 6 Frquencies of inuersion heterozygotes in Brazilian populations of D. kikkawai ~~ ~ Locality Time N n n/N Total Port0 Alegre, RGS March 1952 2 February1955 20 3 0.15 0.14 Florianopolis, SC July 1954 16 3 0.19 September 1955 19 4 0.21 0.20 Gaspar. SC June 1952 7 1 0.14 July 1954 2 1 0.50 0.22 Itajai. SC November 1952 3 2 0.67 June 1953 5 2 0.40 July 1954 9 2 0.22 0.35 Morretes. Pr November 1951 10 August 1953 9 1 0.11 October 1953 2 April 1954 38 5 0.13 0.10 Paranagui, Pr March 1952 10 1 0.10 September 1952 58 3 0.05 October 1953 11 1 0.09 ApriI 1954 2 0.06 Antonina. Pr November 1951 12 2 0.17 March 1952 2 1 0.50 0.21 Slo Paulo. SP May 1954 14 4 0.29 0.29 Lins. SP April-May 1954 19 Belo Horizonte, MG February1953 2 August 1953 6 1 0.1 7 0.13 Others’ 4 Total 282 37 0.13 0.13 N-nuniber of females which have been analysed; n-number of larvae showing the inversion (see METHODS) ; Total- mean number of inversions per larva in the total for each locality; all the collections at one locality were statistically homogeneous. * Others refers to Goiania, Go (September 1953) and Irati, Pr (April 1952). DISCUSSION Color polymorphism: 1. Geographical heterogeneity. The distribution range which has been investigated seems to be sufficiently large and the differences verified along this range sufficiently striking to permit the conclusion that the frequency of the light form varies in the different localities of the area which has been surveyed. No cline is apparent, however. Rather, a sharp and nam limit between two main subareas seems to emerge from the consideration of the whole information. The southmost area (here called region A) is characterized by a mean frequency of the light form around 0.65; the central area (region B) is characterized by higher frequencies of the light form (average around 0.85). Although these two areas seem to present a clear-cut separation, a certain hetero- geneity has been found within each one. We do not have any explanation for two Y-aces” of D. kikkawai being separated by a so narrow and sharp limit as shown by the data (see also Figures 1 and 3). 798 N. FREIRE-MAIA AND A. FREIRE-MAIA 152 80851 l4Y 70 3. 5.06 65- 60- 55. 50 .. 7: 45- ir 40j:::::::::::::" 0 200 400 600 800 1,000 1,200 1,400 KM. FIGURE3.-Frequencies of the light form in different Brazilian populations. Abcissa, the distance in kilometers from Purto Alegre. Ordinate, the frequency of the light form. Dots repre- sent the pled data of males and females (cf. Table 4). Female symbols represent female data (cf. Table 1). Male symbols represent male data (cf. Table 3). 1. Porto Alegre; 2. Florian6polis; 3. Blumenau; 4. Gaspar; 5. Ascurra; 6. Itajai; 7. Paranagu6; 8. Morretes; 9. Antonina; IO. Londrina; 11. S. Paulo; 12. Presidente Prudente; 13. Lins; 19. Belo Horizonte; 15. Goilnia. The distance between Paranagu6 (the northmost town belonging to region A) and Antonina (one of the two closest tolwns of region B) is only 20 km in a straight line on the Paranagu6 Bay. The distance between Paranagu6 and Mor- retes (the second closest town of region B) is only 30 km. The altitude of the three localities is about the same. These facts-although puzzling-are very interesting because they show that even relatively neighboring domestic populations of organisms capable of being easily carried by man (mainly through fruit transportation, an intense activity in the region of ParanaguA, Morretes and Antonina) may undergo racial differ- entiation, probably owing to a differential action of natural selection. This con- clusion has been reached for the first time about 9 years ago (FREIRE-MAIAand FREIRE-MAIA1955). It is interesting to note that the frequency of D. kikkawai among collected domestic species of Drosophila is about the same (8 percent) both in region A and B. 2. Seasonal heterogeneity. Some suggestion has been found of different fre- quencies of light females in the collections made in different seasons of the year, but there is neither a seasonal consistency in the same locality nor a constancy of findings in different localities. Our impression is that the variations which have been found may really represent the result of local variations on the environ- mental conditions without any direct connection with cyclic climate situations. POLYMORPHISM IN D. kikkawi 799 For domestic species which depend largely on the amount, type and conditions of the food that is offered by man, the occurrence of some micro-variables within the environment seems rather possible. The fact that our findings do not show any evidence of being either cyclic within the same locality or consistent with findings from other places points to this hypothesis. 3. Hardy-Weinberg distribution. Our whole data show clearly that the geno- types resulting from the combination of the two alleles of the major gene respon- sible for this color polymorphism are present in natural populations with fre- quencies in accordance with the Hardy-Weinberg Law. No exception has been found among the large number of samples which have been tested with laboratory strains. It is interesting that the results obtained with light females inseminated in nature show, on the whole, apparent examples of Hardy-Weinberg distribu- tions, as if each female were inseminated practically only once and as if no great differences exist in the degrees of sexual activity of males of the three genotypes. The only exception (found in Paranaguh) is due to a highly significant apparent excess of heterozygotes. Comparisons have been made between the distribution of the three main geno- types as determined by analyzing the off spring of collected males crossed to light females from laboratory strains (Table 3), and the distribution found by ana- lyzing light females inseminated in nature (Table 5). Three of the six compari- sons show definitely nonsignificant differences, but the other three give P- values close to 0.05. The only consistent deviation found in all the six samples of Table 5 (i.e., in those with significant deviations as well as in those with nonsignificant ones) is that heterozygotes have been found in excess. The significance of these findings is, however, only hypothetical because we have assumed that a light female giving rise to light and dark offspring has been inseminated by one hetero- zygous male. 4. Genetic Load. Experimental data (FREIRE-MAIA1964a,b) showed that the viability of heterozygotes is higher than that of both types of homozygotes. the lowest of the three being the viability of the dark homozygote. This may represent an example of overdominance, but no proof for this working hypothesis is avail- able. As pointed out by several authors (beginning with HALDANE1937), a genetic load (in the sense of CROW1958) is expected to be operating in the population. This means that the mean fitness of the population is lower than the fitness of its best genotype (Cc). This lowering may be produced by increased mortality, decreased fertility, etc., all leading to an impairment of the mean fitness of the population as compared with the optimum genotype. In an experiment already described (FREIRE-MAIA1964a), the expressed genetic load has been estimated as about 0.27, i.e.,under the conditions of the experiment, the mean fitness of the three genotypes was about 27 percent lower than the fitness of the heterozygote. Since this lowering was due to an increasing mortality rate, part of the mortality rate of the population was really due to the genetic load under consideration. It is clearly seen, contrary to the opinion of some authors, that this is a useful con- cept to describe the price the population pays for being polymorphic. This is not, 800 N. FREIRE-MAIA AND A. FREIRE-MAIA however, the place to discuss the validity of the theory of genetic load. The reader is referred to papers by LI (1963a,b,c), SANGHVI(1963) , LEVENE(1963), CROW (1963) and FREIRE-~IA(1964~). It seems clear that only under very special conditions may a species support the action of a number of loads of this category. For a discussion of this problem, see HALDANE(1937), HIRAIZUMIand CROW(1960), MULLERand FALK(1961 ) , and FALK(1961). Chromosomal polymorphism: For differences verified within the localities, all the P- values exceed 0.05 (cf. Table 6). For the differences among the 11 groups of data (i.e., ten localities and “Others”), x2 = 20.56, d.f. = 10, P<0.05. The largest contribution to this x2 is given by Itajai (x2= 7.34), where a relatively high frequency (0.35) of the inversion has been found against values ranging from 0.06 (Paranaguh) to 0.29 (S. Paulo). Since the number of strains investi- gated in each locality is generally low (for instance, only 17 in Itajai) and the level of significance used is 0.05, it seems to be more reasonable to assume, as a working hypothesis, that the frequencies of inversion heterozygotes in the Bra- zilian populations of D.kikkawai which have been analysed are about the same (around 0.13 inversion per larva). The localities from which samples have been investigated (Table 6) extend from the southmost Brazilian State (Rio Grande do Sul) to a central one (Minas Gerais) . There is no apparent reason why the frequency of inversion heterozygotes in Itajai (at about one third of the range) would be higher than in the others. The action of purely local conditions capable of leading to a relatively high frequency of heterozygotes for that inversion is not impossible, hmever. As pointed out regarding color polymorphism, the habitat of domestic species is largely dependent on man’s action. We are grateful to a number of friends and colleagues who collected or helped us collect some of the samples which have been analysed. It is a pleasure to refer specially to PROFFSSORS JESUSS. MOURE,C. PAVAN,0. FROTA-PESSOA,CLAUDIO FROELICH, JosB PELLEGRINO,A. R. COR- DEIRO, ISMAELFABRICIO ZANARDINI, and DR. JosB PORTUGALFILHO. Our thanks are also due to PROFESSORWARWICK E. KERRfor reading the manuscript. SUMMARY AND CONCLUSIONS D. kikkawai presents three types of polymorphisms: a color polymorphism associated with a sexual dimorphism; a chromosomal polymorphism expressed in a paracentric inversion; and a mitotic chromosome heteromorphism. Frequencies of the color forms from natural populations of several localities in south, east and center-west Brazil have been determined. The southmost localities have a mean frequency (around 0.65) of the light form lower than elsewhere (around 0.85). No gradient, but a sharp limit between the two main groups, has been observed. This limit is located between two neighboring towns. No reason for such a phenomenon is apparent. It shows, however, that domestic populations of Drosophila living in neighboring towns may undergo racial differentiation. This is probably due to the differential action of natural selection in the two towns, POLYMORPHISM IN D.kikkawai 801 The results of different collections made in the same locality do not show significant heterogeneity. The pooled data, when classified according to the month of collection, give some suggestion of heterogeneity, There is not, however, either a seasonal consistency in the same locality or a constancy of findings in different localities. Therefore, it is assumed that the heterogeneity found may result from local variations in environment without any direct connection with cyclic climate situations. For a domestic species, which depends largely on the amount, type and conditions of the food that is offered by man, the occurrence of micro-variables within the environment is rather likely. The observation of natural populations suggests, and experimental data clearly reveal, that heterozygous (Cc) flies have the highest adaptive value, light ones (cc) are intermediate, and homozygous dark (CC) are least fit. In natural as well as in experimental populations, however, frequencies of the three genotypes con- form to the Hardy-Weinberg formula. This indicates that, in spite of the fact that heterozygotes possess the highest total fitness, selective forces seem to favor both classes of homozygotes in at least one of the components of that complex biological parameter. All available data lead to the conclusion that the existence of the two color forms in natural populations is under the control of selective forces. These forces seem to operate with diverse intensities in different places. Besides the advantages conferred on the species by this polymorphism, a genetic load develops as its consequence. The fact that the genotypes responsible conform to the Hardy- Weinberg Law shows that, ceteris paribus, the expressed genetic load is here larger than it would be if negative selection was operating only against homo- zygotes. The only paracentric inversion which has been found in natural populations of D.kikkawai is present with about the same frequencies (around 0.13) in all the samples analysed, with only one possible exception. No quantitative infor- mation is available on the presence, in natural populations, of the two chromo- some forms responsible for the heteromorphism of chromosome IV. LITERATURE CITED CROW,J. F., 1958 Some possibilities for measuring selection intensities in man. Human Biol. 30: 1-13 - 1963 Genetic load: Three views. 2. The concept of genetic load: a reply. Am. J. 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