Local and Seasonal Variations of Lethal Frequencies. Natural Populations of Drosophila melanogaster. Sumlo MINAMORI and Yoshlnori

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1 JAP. JOUR. GENET. Vol. 38. No. 4 : (1964) Local and Seasonal Variations of Lethal Frequencies Natural Populations of Drosophila melanogaster in Sumlo MINAMORI and Yoshlnori SAITO Zoological Laboratory, Faculty of Science, Hiroshima University Hiroshima, Japan Receined November 6, 1963 Genetic structure of natural populations may be the result of interactions between the mode of breeding and the habitat. The mode of breeding in a Drosophila natural pepulation can be determined to some extent, by studying the deleterious genes contained in the population. The population size may be estimated when the equilibrium frequency and the rate of allelism of the lethal genes are known. The effective number of populations in Shizuoka or Hiroshima area have been estimated to be around 1,000 by using those parameters (Oshima and Kitagawa 1961, Minamori, Ikemoto and Fukaya 1962). The number indicates that these populations are apparently small, which seems to be brought about by a contraction of the population size in winter season in those areas. If so, frequencies and rates of allelism of the lethal genes might be changed with the expansion of population size with the advancing breeding season. A seasonal change in lethal gene frequency was observed first by Dubinin (1946) in Russian populations of Drosophila melanogaster. The change was also studied by Ives (1954), Goldschmidt et al. (1955), Band, Sheppard and Ives (1959), Hiraizumi and Crow (1960) and Karlik and Sperlich (1962). Although they observed the frequency of lethal genes and also their allelic rates continuously for a fairly long period, no remarkable change could be detected. The main aim of the present study is to find out whether seasonal changes appear in the Hiroshima populations. Ranges of territory of a population may be estimated, when allelic rates within or between populations are known, as studied by Wright, Dobzhansky and Hovanitz (1942) in Drosophila pseudoobscura populations. Another aim was to determine the ranges in the area. In the present paper, lethal frequencies and allelic rates in natural Hiroshima populations of D. melanogaster in different habitats, seasons and years, are described and the mode of breeding of the fly in the area is determined.

2 LOCAL AND SEASONAL VARIATIONS OF LETHAL FREQUENCIES 291 Material and Method F1 ies were collected at one or two places in Hiroshima City, Kure City Shiwa Town in 1961 and The distances between these localities or stations shown in Figure 1. All traps were set at indoor places, viz, two grocery stores, pickle factories and a storehouse of a farmer, as shown in Table 1. and are two Fig. 1. Distances among stations set in three localities. TTY Table 1. Places of Collection Summer samples of 1961 were collected at three stations set in Hiroshima City and in Shiwa Town in late June. Autumn samples were collected at the same station and other two stations set in Kure City about 70 days after, in early October. At the earlier season the population size seemed to reach the first peak and at the later season it appeared to expand to the maximum (Figure 2). In 1962, a summer sample was collected only from the Befu Station set in Shiwa Town and the autumn collection was made after about 80 days at the Tera Station set in Hiroshima City and the Befu Stations. A second chromosome was isolated from a single male fly collected from natural populations by the marked inversion method. In the F3 generation after mating, the wild chromosome was duplicated. When non-cy flies did not appear in the F3 generation, the isolated second chromosome was considered as carrying a lethal gene and when the number of non-cy flies was less than 16.7 per cent of all emerged flies, the

3 292 S. MINAMORI and Y. SAIT0 second chromosome was considered as carrying semilethal gene. Flies were cultured at 25 C in a glass tube containing cornmeal-molasses-agar and yeast medium. Results 1. Seasonal changes in population size In Hiroshima City, fruit flies begin to appear usually in early July and disappear in December. The seasonal changes in population size of the Midori Station set in Hiroshima City was examined in Six cubic cans of 10 X 10 X 10 cm3 volume provided with banana-yeast medium were set up in the grocery. The next day these cans were brought back to the laboratory and the number of collected flies was counted. These traps were set every third week from early July to late December. The results obtained are shown in Figure 2. The temperature changes in Hiroshima City during the period are also shown in the figure; they are taken from the Report of the Hiroshima Climatological Station. In the figure the ordinates indicate the number of collected flies per can. Fig 2. Seasonal change in population minimum temperatures, Below size. Upper maximum and number of flies collected. In Hiroshima City the flies have not yet been collected earlier than May since The earliest collection could be made on June 24th of According to the survey in 1960, flies were collected for the first time in mid July and their number increased and reached to the first peak in early August. This peak coincided roughly with the temperature maximum of the year. Then, the number of flies collected

4 LOCAL AND SEASONAL VARIATIONS OF LETHAL FREQUENCIES 293 decreased and increased again in early September. Daily maximum temperature at the turning period was about 25 C. The maximum number of flies was obtained in early October, when the daily maximum temperature was about 20 C. In the middle of December, flies could not be collected. Such a bimodal curve in the population size indicates that the natural population is suppressed by high temperature in summer and severely handicapped by low temperature in winter. To estimate the number of generations elapsed during the two collection periods in the natural populations, some wild stocks were cultured in the laboratory room. The first emerging five pairs of males and females were transferred to a new glass tube. Room temperatures were recorded simultaneously. The experiment was continued from early March to early December of 1961, since flies could not emerge from late December to late February. The results obtained are shown in Figure 3. About 18 generations elapsed in this period. According to the result obtained, it may be concluded that about seven consecutive generations elapsed in the natural populations between the first and second collections. Fig. 3. Generations elapsed in a maximum and minimum year under the laboratory temperatures in the course temperature. of generation. Upper started point of the experiment. 2. Frequencies of lethal chromosomes Total of 1901 wild second chromosomes isolated from summer and autumn collections at five stations in 1961 and 1962 were duplicated and tested. The results are shown in Table 2. The total frequencies of lethal and semilethal chromosomes in each station ranged from 2.2 per cent (the Befu Station in summer of 1962) to 20.7 per cent (the Midori Station in autumn of 1961). In the former station, none of lethal chromosome was detected. The pooled frequency was about 11.6 per cent. This value approaches closely that reported in the previous paper (Minamori and Azuma 1962). Frequencies of chromosomes bearing semilethal genes were generally lower than of those bearing lethal genes through these populations. However, the former exceeded, on the average, the latter by more than 50 per cent. The result is slightly different from that for the Shizuoka populations reported by Oshima and Kitagawa (1960).

5 294 S. MINAMORI and Y. SAIT0 Table 2. Frequencies of chromosomes bearing lethal and semilethal genes a) Variations in the lethal frequency of different populations Variations in the lethal frequency were observable among populations in different stations. The total frequencies of lethal and semilethal chromosomes in several populations located at different stations and habitats were compared with each other (Table 3). The frequency in the population of the Befu Station was notably less in cotrast to those of other stations in the other localities. The differences between the Befu Station and each of the two stations set in Hiroshima City were highly signifi- Table 3. Differences in the combined frequency of bearing chromosomes between populations lethal from and semilethal different places genes

6 LOCAL AND SEASONAL VARIATIONS OF LETHAL FREQUENCIES 295 cant. The frequencies obtained in those two stations set in the same locality in both Hiroshima and Kure City were not significantly different from each other. The frequencies obtained in those two stations set in groceries were significantly different, but between the two stations set in pickle factories were similar to each other. These results seem to indicate that the frequency of lethal and semilethal chromosomes in a population is not only affected by the habitat of populations but also by some other factors. b) Seasonal changes in the lethal frequency Seasonal changes in the lethal frequency were examined with respect to summer and autumn collections from Midori, Tera and Befu Stations in 1961 and from the last station in Both frequencies of lethal and semilethal chromosomes were consistently increased as shown in Table 4. The increment in the Befu Station was less than those in the other stations. The lethal frequency in this station was always found to be low in the successive five sueveys. If such a parallelism between the lethal frequency and the increment is valid, the effective number of the population might be very small throughout the year. The pooled increment of the four surveys of lethal chromosomes was 2.53 per cent and that of semilethal chromosomes was 0.99 per cent. The combined increment of the lethal and semilethal chromosomes was 3.52 per cent and it was statistically significant. According to the assumption that mutation rate is 105 per locus, the number of lethal producing loci on the second chromosome is 500 and as seven generations passed during the two seasons, the increment of lethal genes in these populations should be 3.5 per cent (10.5 X 500 X 7). This value appears to be higher in contrast to that abtained in the survey. However, if we take into consideration the following parameters; selection coefficient of lethal heterozygotes, Poisson distribution of lethal genes in the chromoseome, effective number of populations during the two collections, the calculated value should become lower. Therefore, the increment obtained in the survey may be considered to be close to the expected one. Table 4. Seasonal changes in lethal frequencies (lethal + semilethal, % ) c) Annual changes in The collection of flies the lethal frequency from the Midori Station has been carried out since 1959

7 296 S, MINAMORI and Y. SAIT0 and from the Befu Station since The experimental results obtained in 1959 and 1960 were reported in the previous paper (Minamori and Azuma 1962). The lethal frequency in populations of these stations seemed to be fixed without remarkable fluctuations. Three summer collections of the Midori Station in 1959, 1960 and 1961, three autumn collections of the Befu Station in 1960, 1961 and 1962, and two autumn collections of the Tera Station in 1961 and 1962 were statistically homogeneous in each station. These results indicate that the lethal frequencies in these populations are gradually increased during the breeding season and decreased during the dormant season. Such a seasonal change seems to be repeated within a definite range of the fluctuation in each year. 3. Visible mutants Several kinds of visible mutants were detected in the present experiment. Among them two kinds of eye color mutants were found in samples collected in One was brown and the others were Lobelike mutants. The former was identified by crossing it with a brown strain cultured in the laboratory. The latter was dominant, though the penetrance was somewhat varied. In the Tera Station, twelve chromosomes bearing the brown gene were detected in a summer collection, while only one chromosome was isolated from an autumn collection (Table 5). The Lobelike gene was detected on four chromosomes in the autumn collection from the Midori Station and on one chromosome in that from the Kegoya Station. In the other stations, none of such eye color mutants was detected. The deviation in the appearance of these mutants among these populations seems to indicate that migration of flies among stations scarcely occurs. This surmise would be also supported by the results obtained in allelism test described below. 4. Allelic rates between lethal genes a) Allelic rates within each population Table 5, Visible eye color mutants from natural populations extracted in 1961 Allelism tests on lethal genes on the chromosomes extracted from the same population were performed and the results are shown in Table 6. Allelic rates in these populations were distributed from zero to 60 per cent and the average was about 7 per cent. Thus a variation in the rate in different populations was detected. The allelic rates in the Midori Station in 1961 were quite similar to those in the two stations set in Kure City, but higher in the Tera Station set in the same city. The difference between allelic rates at the Midori and Tera Station in 1961 was statistically significant (0.05> P > 0.02). The population of the Befu Station seemed to differ from those of the other stations, because allelic rates fluctuated markedly with

8 LOCAL AND SEASONAL VARIATIONS OF LETHAL FREQUENCIES 297 Table 6, Allelic rates within population seasons or years. Such fluctuations might be caused by sampling errors. The allelic rates in these populations may be decreased in the breeding season. In fact, the difference between the average allelic rates in summer and autumn populations at the Midori, Tera and Befu stations in 1961 was 2.26 per cent. However, this difference was not statistically significant. The allelic rates at the Midori Station obtained in 1959, 1960 and 1961 were very similar to each other. But the allelic rates in the autumn collection of Tera Station in 1962 were much higher than those obtained in the same season of the previous year and the difference between them was highly significant (P <0.01). This change may be supposed to be due to a generation of heterotic lethal genes in that population (see Section c). If these heterotic alleles are left out of consideration, the rate in 1962 would become zero, rather close to the rate obtained in A similar repeated appearance of a heterotic allele was observed in the population of the Midori Station in These points will be described below in detail. b) Allelic rates of lethal genes extracted from different populations Allelic rates between lethal genes on chromosomes extracted from different stations in the same season of the same year are shown in Table 7. In the allelism test, one representative chromosome was used when a lot of chromosomes bearing allelic genes were obtained, except for the tests between populations from Shizuoka and from Hiroshima areas. The allelic rates between lethal genes extracted from different stations were much lower than those in the same population. This fact indicates that migration of flies

9 298 S. MINAMORI and Y. SAIT0 Table 7. Allelic rates between populations from di fferent places in the same season among these stations would occur very seldom, even between stations set in the same city. The results obtained in intra- and interpopulational allelism tests suggest that the Tera and the Midori populations may belong to independent breeding units. If

10 LOCAL AND SEASONAL VARIATIONS OF LETHAL FREQUENCIES 299 so, the territory of each population may be assumed to be narrower than the area of a circle with 1.75 km radius, because the two stations were apart by 3.5 km between them. The allelic rate between lethal genes extracted from natural populations of Hiroshima and of Shizuoka areas was 0.23 per cent (Table 7, C). This value is closed to that expected when the allelic lethal genes arise independently. Therefore, these two populations are regarded to be completely isolated from each other. The allelic rates betweem lethal genes extracted from summer and autumn populations of the same station in the same year (A), and between lethal genes extracted from populations of the same station in the same season of two consecutive years (B) are shown in Table 8. The former allelic rates were lower than intrapopulational allelic rates and rather close to those between stations. When the allelic rate between all lethal chromosomes was obtained, it became higher as given in the parenthesis of Table 8. It is close to those obtained in intrapopulational tests. This phenomenon indicates that some lethal genes might persist during two seasons. On the other hand, none of allelic genes could be found in all lethal genes isolated from the same station in two successive years at all three stations. This result is in sharp contrast to that obtained in Shizuoka populations in which a persistence of lethals was detected (Oshima 1962). Table 8. Allelic rates between populations from different seasons or years in the same station 5. Existence of cis-formed double lethal chromosomes in natural populations Three kinds of chromosomes bearing two lethal genes were detected by allelism

11 300 S. MINAMORI and Y. SAIT0 tests. The one kind was extracted from summer collections of the Midori Station in 1961, and the other two kinds were extracted from autumn collections of the Tera Station in In the former station five chromosomes bearing allelic lethal gene were detected in the summer collections. Four chromosomes among them have been maintained in cy-balanced system and they were used for the allelism tests with the autumn collections of the same station in the same year. The lethal gene carried by them was proved to be allelic to lethal genes located on the five chromosomes extracted from autumn collections. The half diallele cross by using all nine lethal strains was performed and it was shown that two groups of three chromosomes had a non-allelic lethal gene. The relationship is shown in Table 9. This result could be consistently interpreted by assuming that three chromosomes (20, 130, 151) in the summer collections have, at least, two lethal genes as shown in Figure 4. If this assumption is correct, the cis-formed double lethal heterozygotes could be considered to be maintained and these two lethal genes to be separated by recombination, each of them was found in the autumn population. Table 9. Allelic relationship among lethal genes extracted from the Midori Station in 1961 (Q allele) Figure 4. Double lethals and their alleles. The results obtained in allelism test on the other two kinds of double lethal chromosomes extracted from the Tera Station in autumn of 1962 are shown in Table 10. These two chromosomes (23, 13) bearing double lethal genes were recognized to have one common allelic lethal gene. The relationship is shown in Figure 5. The exceedingly higher allelic rate in the Tera Station in autumn of 1962 is considered to be caused by the increase of these three kinds of lethal genes. A heterotic viability of the cis-formed double lethal heterozygotes obtained in the Midori Station (ab) was reported by Oshima (1963). Relative viabilities of the cd

12 LOCAL AND SEASONAL VARIATIONS OF LETHAL FREQUENCIES 301 Table 10. Allel from is relationship among the Tera Station in lethal 1962 (0 genes extracted allele) Table 11. Relative viabilities of lethal heterozygotes. (Viability of Cy/Pm fly = ) Figure 5. Double lethals their alleles. and and the de double lethal heterozygous flies and of each single lethal heterozygotes were estimated and compared with the viability of normal heterozygotes by using Cy/Pm technique. The results are shown in Table 11. As shown in the table, the

13 302 S. MINAMORI and Y. SAITO viability of double lethal heterozygotes was superior to that of separated single lethal heterozygotes, and even to that of normal heterozygotes. The superiority of the double lethal heterozygotes was recognized to be statistically highly significant (P 0.01). The viability of single lethal heterozygotes was slightly less than that of normal heterozygotes, but the difference was not significant. Among c, d and e single lethal heterozygotes, +/d was recognized to be significantly superior in viability to the other two (0.05 > P > 0.01, P < 0.01). Discussion The lethal frequency in natural populations of D. melanogaster in some localities in Japan was shown by Oshima and ourselves to be as low as in Korean populations (Paik 1960) or Russian populations (Dubinin 1946) and lower than in American populations (Ives 1944, 1954, Band and Ives 1961, Hiraizumi and Crow 1960, Seto 1961) and in Mediterranean populations (Goldschmidt et al. 1955, Dawood 1961, Karlik and Sperlich 1962). According to Prout (1954), the equilibrium frequency of lethal chromosomes in random mating expeimental populations was about 26 per cent. If the equilibrium frequency in these surveyed populations, when the population size is infinite, is assumed to be close to that value, the obtained frequencies (5.56 per cent in the summer populations, average) is much lower. Such an unsaturated content might permit a rapid accumulation of lethal genes in populations whose size becomes large. The low frequency of lethals in these populations is considered to be not caused either by low mutation rate or low viability of lethal heterozygotes, but by the small number in effective size of these populations at some period of the breeding cycle. That frequency may also be brought about by direct selection of winter cold or summer heat against lethal heterozygotes as assumed by Band et al. (1959). At present, it is obscure whether or not such a mechanism plays some role in these populations. The small size of these Japanese populations is also considered by a fact of the biased distribution of visible mutants among populations or by the results obtained in intra- and interpopulational allelism tests. Two populations, living 3.5 km apart from each other, revealed to be nearly independent in their breeding. By further studies the territory of each population might be found to be narrower than deduced from the above result. It is certain that these small populations are isolated, though not completely, from each other, since in five stations they were discriminated from each other either by the difference in lethal frequencies and allelic rates or the difference of visible mutants. In Hiroshima area, many such populations seem to be distributed in a rather narrow space, and to mould each its genetic structure. The seasonal changes in the lethal frequency and allelic rate seem to be repeated

14 LOCAL AND SEASONAL VARIATIONS OF LETHAL FREQUENCIES 303 every year in each station without any remarkable fluctuation, because the frequencies and the rates of populations in each station in the same season were not changed in the consecutive years. This fact suggests that the effective number of populations in each station at the initial generation in the breeding cycle might be similar every year. If so, all populations might have each a specific and rather constant effective number. As already mentioned, some heterotic cis-formed double lethal genes, such as ab, cd and de, were contained in these populations. Such lethals may be propagated rapidly by their heterotic nature and the lethal frequency and allelic rate of the population would be increased. Therefore a great care is necessary for the deduction based on the effective number estimated directly from the obtained lethal chromosome frequency and allelic rate. At any rate, it is apparent that the population of each station starts with a small number of flies. However, it is unknown whether the original flies starting each population are immigrant flies or survived ones in a place. Concerning this point, some experiments and examinotions are undertaken. By our experiment, most strains of D, melanogaster revealed to be killed within two days at 0 C. Furthermore we could not keep alive the flies in a culture bottle at the Befu Station during the winter of Such results seem to support rather the probability of immigration of flies to the station from their hibernating places. This deduction is also justified by the fact that none of lethal genes were, as described, observed to persist over one winter season in three stations, though the lethal frequency and the allelic rate in populations of successive years seemed to be unchanged. It is apparent that the breeding mode affects the genetic structure of population of the fly, as discussed above. However, extensive ecological studies are necessary to get more exact knowledge on the relationship. Summary Frequency of the second chromosomes bearing lethal gene (or genes) in natural populations was examined in five stations, in summer and autumn seasons and in successive years in Hiroshima Prefecture. The combined frequencies of lethal and semilethal chromosomes varied with the stations, seasons or years from 2.2 per cent to 20.7 per cent. The allelic rates within a population ranged from zero to 60 per cent. These results suggest that these populations were small in size. From the results of allelic rates, the narrowest territory of a breeding unit was estimated. The frequency of lethal chromosomes was revealed to be increased by 2.53 per cent and that of semilethal ones by 0.99 per cent from summer to autumn, and about seven generations may elapse in natural populations during this period. The combined increment of lethal and semilethal chromosomes was statistically significant. The allelic rate was observed to be decreased by 2.26 per cent during the two seasons. In spite of

15 304 S. MINAMORI and Y. SAIT0 such an accumulation of lethal genes or decrease of allelic rate in the breeding season, any remarkable changes in the frequency or rate were not observed in the same season of successive years. Therefore, the seasonal changes in lethal frequency and the allelic rate seem to be repeated from year to year with the same fluctuation. Based on these results, the breeding mode of natural populations of D. melanogaster distributed in Hiroshima area was discussed. Acknowledgment The authors wish to thank Dr. C. Oshima, National Institute of Genetics, for his valuable criticism, and Mr. M. Azuma for his collaboration in this work. Literature Cited Band, H. T, and P. T. Ives 1961 Correlated changes in environment and lethal frequency in a natural population of Drosophila melanogaster. Proc. Nat. Acad. Sci. 47: