DROSOPHILA MERCATORUM

Transcription

1 SELECTION FOR MATING RELUCTANCE IN FEMALES OF A DIPLOID PARTHENOGENETIC STRAIN OF DROSOPHILA MERCATORUM HIROSHI IKEDAl AND HAMPTON L. CARSON2 Department of Biology, Washington University, St. Louis, Missouri Manuscript received March 19, 1973 Revised copy received June U), 1973 ABSTRACT A diploid Parthenogenetic strain of Drosophila mercatorum was outcrossed to produce genetic variance among the impatemate female offspring. Selection experiments were carried out for reluctance of the parthenogenetic females to mate. After only two cycles of selection, a parthenogenetic strain which is significantly less receptive to males from three different bisexual strains was obtained. It was also found that there is some degree of sexual isolation among the three bisexual strains used. The results support the idea that selection can render a newly produced diploid parthenogenetic strain behaviorally different from its bisexual ancestor. This appears to provide a framework which can explain the natural coexistence of diploid bisexual and diploid parthenogenetic biotypes in some species of insects. ARTHENOGENESIS, as a substitute for syngamy, has evolved independently 'as a mode of reproduction in many insects. A commonly observed type is thelytoky, wherein unmated females essentially produce only daughters and the male sex is effectively eliminated from participation in reproduction. Cases where a diploid thelytokous form coexists with its evident diploid bisexual ancestor are of special interest from the point of view of microevolution. Thus, females of the parthenogenetic biotype must somehow have acquired a reluctance to mate with males of the bisexual form. Such a situation exists naturally, for example, in the psychid moth Solenobia triquetrella in Switzerland (SEILER 1961 ). Reproductive isolation of the diploid parthenogenetic form in nature seems to be complete although females of this biotype can be crossed to males of the bisexual form in the laboratory (SEILER 1966,1968). The present paper reports experimental studies of a comparable system in Drosophila. In nature, D. mercatorum appears to reproduce, as do most species of the genus, wholly by ordinary bisexual means. Nevertheless, building on a very low rate of facultative parthenogenesis, CARSON (1967) was able to synthesize by artificial selection, a number of vigorous diploid parthenogenetic laboratory strains which reproduce without males. The present paper examines the sexual behavior of females derived from one of these strains and reports on the Present address: Biological Institute, Ehime University, Matsuyama, Ehime 790, Japan. a Present address: Department of Genetics, University of Hawaii, Honolulu, Hawaii Genetics 75: November, 1973.

2 542 H. IKEDA AND H. L. CARSON outcome of a program of artificial selection of females of this strain for increased reluctance to mate. This reluctance is displayed in the presence of males from any one of three different bisexual strains. MATERIALS AND METHODS Strains used: All of the strains used in the present study belong to Drosophila mercatorum. An original parthenogenetic strain, RSB-7-Im, used for the selection experiments was obtained from a cross of the bisexual R (Rochester) and S (El Salvador) strains and has been maintained parthenogenetically since 19f3 with a parthenogenetic rate of 3.2% (CARSON 1967). Recent work (CARSON 1973) shows that 213% of the impaternate offspring of RSB-7-Im are produced by pronuclear fusion of two meiotic products. The remainder arise by meiotic doubling of a haploid nucleus. To introduce genetic variability into a parthenogenetic strain, males from a Bridge strain having a high degree of parthenogenesis in its genetic background were used. The Bridge system was outlined by CARSON (1967) and CARSON and SNYDER (1972). All strains used are summarized in Table 1. The generation of genetic variability: In parthenogenetic stocks of D. mercatorum, most progeny result from post-meiotic doubling. Thus, after a few generations, all such stocks become largely homozygous (CARSON, WEI and NIFDERCORN 1969). In order to generate genetic variability, females of RSB-7-Im were outcrossed to males of a Bridge strain, OB-2Br (see Figure 1). About 300 F, females from this cross were collected as virgins and were placed in food vials without males to reproduce parthenogenetically. During this phase, recombinations and immediate fixation of many different genotypes in the homozygous state are expected. Procedures of the first cycle of selection. Five hundred of the genetically variable impatemate females emerging within 24 hours were isolated and aged for four days. To select females which are reluctant to mate with bisexual males, groups of twenty females aged for four days were placed with twenty, seven-day-old males from each of the R and S strains in each mating vial. Mated pairs were aspirated out as they formed and the strain to which the male belonged was recorded before discarding. The females unmated during the observation period (two hours) were collected with an aspirator and stored in food vials so that subsequent observations could be made on them. This series of procedures was called a mass screening. Next evening, those females which had not previously mated ( non-maters ) were introduced again into mating vials with males and treated in the same manner mentioned above. Mass screenings were repeated seven times through seven days. Observations of mating speed and sexual isolation were carried out TABLE 1 Laboratory strains of D. mercatorum used in the experiments Bisexual strains* R : Rochester, New York, collected in Oct., 1957 S : La Palma, El Salvador, collected in Aug., : Pupukea, Oahu, Hawaii, collected in July, 1963 Parthenogenetic strain RSB-7-Im: established in Dec., 1964 by H. L. CARSON; parthenogenetic rate 3.2% (see CARSON 1967) Bridge strains OB-2-Br : originally derived from R, S and 0 strains (see CARSON 1967) 0-3-Br : originally derived solely from the bisexual strain 0 mentioned above (see CARSON 1967) * These three strains are distinguishable from each other by the degree of coloration of the abdomen.

3 MATING RELUCTANCE IN D. mercatorum 543 Cycle 1 PARTHENOGENETIC FEMALES X BRIDGE MALES (RSB-7-Im) I (OB-2-Br) F~ FEMALES F, MALES (vi rg i n) (d i scard) I MPATERNATE FEMALES (500) select single non-mating females strains from single impaternate females + 4, J. + 4, 4 4, TEST OF MATING SPEED' select a slowest mater against wild type males FEMALES (Se1-1 -Im) i UN RE LATE D BRIDGE MALES (0-3 - Br ) FIGURE 1.-Procedures of selection for a parthenogenetic line which shows reluctance to mate with males from bisexual strains in D. mercatorum. after 7 p.m. (late evening) under moderate overhead ceiling light (four 20W fluorescent lamps). The evening hour was selected because HENS- (personal communication) found that D. mercatorum is sexually more active in the evening than during the daytime. Indeed, according to unpublished observations (IILEDA, in preparation), R and S flies of this species which were kept under an artificial dark and light cycle (12 hours: 12 hours) for seven days before mating tests show maximum mating indices (SPIESS, LANGER and SPIESS 1966). The minimum variances occur at the time when the light was turned off in the evening. Such a tendency was not shown in the

4 544 H. IKEDA AND H. L. CARSON daytime or in the morning when the light was turned on. The mating vial was a glass tube measuring 32 mm x 90 mm. The open end of this vial was covered with thick paper which had a small hole. This hole served as a passage for transfer of flies into and out of the vial. Prior to tests, a piece of moistened tissue paper was inserted to raise humidity in the mating vial. Flies used were reared on ordinary cornmeal-agar-molasses food with added yeast in a laboratory room (about 25 ) with natural diurnal lighting. Flies were handled with the aid of an aspirator without etherizstion except for the collection of virgin flies. Females remaining unmated by the end of the seventh day were allowed to lay eggs individually without males to establish parthenogenetic iso-female lines. Lines which produced bisexual offspring were discarded, because this mating was assumed to have gone undetected during mass screenings. Seven of the iso-female lines which consisted of only female offspring were picked at random and allowed to produce parthenogenetic offspring in small mass cultures. Several parthenogenetic generations were required to produce stocks large enough to be tested for mating speed. Tests of mating speed were made with males of R and S strains on each of seven lines by the method to be described later. Mating speed with 0 (Oahu) males was measured only on a previously selected line, in which females were reluctant to mate with both R and S males. The slowest line measured against males of all three different bisexual strains was selected and served as a foundation population for the next cycle of selection. It was designated Sel-I-Im. Procedures of the second cycle: During the course of the first cycle, the following problems arose and were dealt with in successive cycles. First, in the F, of the outcross between parthenogenetic femdes and the Bridge males, two kinds of offspring might be produced. One of them is produced bisexually, and the other is produced parthenogenetically. In order that the latter type of offspring might be distinguished from the former, a Bridge strain, 0-3-Br, marked with a dominant sex-linked gene, sl+ ( spotless-plus ) (see CARSON, WEI and NIFDERCORN 1969 and CARSON and SNYDER 1972), was used in the second cycle. Offspring marked with sl+ were known to be produced bisexually and were then collected as virgins. In the next parthenogenetic generation, however, two kinds of offspring were produced. Most of them are homozygous for either sl+ or sl. Two hundred and sixty females each of sl+ and sl were collected and subjected to mass screenings separately. Second, it seems to be unnecessary to observe for two hours during mass screenings, because about 80% of females mated in the first hour. Accordingly, the observation time was shortened to one hour in the second cycle. Third, the experiment testing interaction in mating between R and S males showed that R males are less active than S males and that the mating activity of R males seems to be strongly depressed by the existence of S males in a same vial (IKEDA 1972). To eliminate this kind of interaction, impaternate females were placed with R males on the fwst day of mass screening and with S males on the second day. This procedure was repeated three times. On the seventh day, females remaining unmated were placed with 0 males to select out females which prefer the latter. Other procedures were the same as those of the first cycle. Control procedures for mms screenings: To investigate the behavior of bisexual females in mass screenings, five kinds of control mass screenings using R and S strains were carried out. Control 1 : twenty, four-day-old R females were placed with twenty, seven-day-old males each of R and S strains in an observation vial. Other procedures were the same as those of the mass screenings of the first cycle. Controls 2 and 5: In Control 2, twenty, four-day-old R females were placed with thirty males of the same strain (R) in a vial in the first day, and next day females remaining unmated were placed with males of an unlike strain (S). Control 5 was made using S females instead of R females in the case of Control 2. Other procedures were the same as those of the second cycle. Controls 3 and 4: These were basically the same as Controls 2 and 5, with the exception that the females were put with unlike males on the first day and then like males on the second day. In each control mass screening, three hundred females were tested in total, except that five hundred females were used in Control 1. Tests of mating speed: Mating speed was measured by the proportion of matings having occurred within a one-hour observation time. Adults emerging within 24 hours were sexed and separated, then aged for seven days in heavily yeasted food vials. Flies were transferred twice into fresh vials during the aging period (on the third and the sixth days after eclosion). In each

5 MATING RELUCTANCE IN D. mercatorum mating vial, ten females of a given strain were placed with twenty males either of R, S, or 0 strain. Pairs mating were removed with an aspirator as soon as they formed. The score of matings were divided into three groups, Period I (0-10 min), Period I1 (10-30 min), and Period I11 (30-60 min). For each of all possible combinations among R, S, 0 and RSB-7-Im strains, twenty replicates were run, e.g., two hundred females were tested, although 10 replicates were performed for the tests made during both the first and the second cycles. Measurements of the degree of sexual isolation: Tests of sexual isolation were made by means of the so-called male multiple choice method for all possible combinations among R, S, 0, RSB-7-Im, Sel-1-Im and Sel-2-Im strains. Flies emerging were sexed and aged with the same manner mentioned in a previous paragraph. Ten virgin females each of two different strains were placed with ten males of one of the bisexual strains in a mating vial. Pairs mating were aspirated out as they formed to prevent males being allowed a second mating, and the type of mating, homo- or heterogamic, was recorded. Observation was carried out for one hour. For each combination, ten replicates were run. An isolation index was obtained on the pooled data by use of the formula presented by STALKER (1942). In combinations between S and parthenogenetic strains, S females were marked by clipping of the left wing, since S females are indistinguishable from parthenogenetic ones. R and 0 flies are distinguishable from each other and also from S and parthenogenetic ones by means of the coloration pattern of the abdomen. Rate of parthenogenesis: The rate of parthenogenesis was measured as the percentage of eggs laid emerging as adults. Twelve virgin females emerging within twenty-four hours were kept on the food enriched by live yeast. On the fifth day after eclosion, females were placed individually in vials containing yeasted food and transferred every other day for ten days. The eggs laid and adults emerging were counted. Strains tested were S, RSB-7-Im, Sel-I-Im, Sel-If-Im, Sel-2-Im and Sel-Zf-Im. 545 RESULTS Mating speed of three bisexual strains and an original unselected Parthenogenetic strain Mating speed of bisexual strains for all possible combinations among R, S, and 0 are summarized in Table 2. The contingency x2 values for group comparisons were obtained on the basis of the number of females mated by the end of one hour and those remaining unmated. When the data shown in Table 2 are rearranged with respect to the male strains, they can be summarized as, R8 : SO>> R?>> 00 S8 : SO>> OO>> R? 08 : SP>> O?>> RP where >> means that the difference in mating speed is significant at the 0.1% level. It is striking that S females were approximately equally receptive to all three kinds of males. On the contrary, R females were extremely reluctant to mate with S males; only 20% mated. 0 females seem to show an intermediate mating speed. It is likely that the female is more important in determining mating speed than the male. When mating speeds are summarized with reference to the male strain, they are significantly different in all six group comparisons. On the other hand, referring to the strain of origin of the female (Table 2), only three out of six comparisons show significant differences. Mating speeds of the unselected parthenogenetic strain, RSB-7-Im, are shown in Table 3. When tested with S males, RSB-7-Im showed the highest mating

6 546 H. IKEDA AND H. L. CARSON TABLE 2 Mating speeds for combinations among three bisexual strains and x* values for group comparisons No. of No. of females Mating females mated in each period Speed Female Male tested I I1 I11 (%) X* R R ** O *** S S R 200 1M Observation period: I, 0-10 min., II,10-30 min., III,30-60 min. ** P < 0.01, *** P < ** 0.31 speed, 82%, which is lower than that of homogamic mating of S, but higher than those of Sa x RP (20.5%) and Sa x 0 0 (72.5%). When tested with R and 0 males, mating speeds are 61% and 62%, respectively. There is no significant difference between them. These values are significantly smaller than those of combinations among bisexual strains in which either R or 0 males were included (P < 0.05, or less). TABLE 3 Mating speeds for combinations between bisexual and parthenogenetic strains and ~2 values for group comparisons No. of No. of females Mating females mated in each period speed Male Female tested I I1 I11 (%) X' R RSB-7-Im *** Sel-I-Im Sel-2-Im S RSB-7-Im Sel-I-Im Sel-2-Im RSB-7-Im Sel-1-Im Sel-2-Im Observation period: I, 0-10 min., 11, ILL30 min., III? 3G60 min. * P < 0.05, *** P < 0.001, 1% Impaternate stram *** 11.58* * * "

7 MATING RELUCTANCE IN D. mercatorum Results of control mass screenings The results of five kinds of control mass screenings are given in the upper part of Table 4. Most of S females mated with males with which they were placed on TABLE 4 Results of control and experimental mass screenings 547 ~~ Control 1) 2) 3) 4) 5) Cycle 1) 2) No. of screenings with malm of R S O (7) (7) (7) (7) No. of females mated with males of R S O Percent of females mated F Im, Impatemate strain. The numbers of screenings where both R and S males were placed with females in a vial are parenthesized. the first day (Controls 4 and 5), whereas only about two-thirds of R females mated for six or seven days screenings (Controls 1, 2, and 3). Contingency x2 tests for the number of R females mated and those remaining unmated were made among Controls 1, 2 and 3. There are significant differences in the mating frequency between Controls 1 and 2 (P < 0.001) and also between Controls 2 and 3 (P < O.Ol), whereas no difference was found between Controls 1 and 3 (P > 0.20). Low mating frequency of R females seems to be due mainly to the fact that the R female is reluctant to accept the S male (Table 2). In addition, in Control 1 some sexual interaction in mating between R and S males seems to occur. It is evident that when R females were placed both with R and S males in a same vial, the mating frequency was decreased as the number of S males increased, whereas such an interaction was not found in the case when S females are used (IKEDA 1972). R females which were placed with S males prior to R males seem to become reluctant to mate even with R males. This effect could account for the difference in mating frequency between Controls 2 and 3. Results of the first cycle of selection The result of the mass screening is shown in Table 4 (lower). After the seventh mass screening, 51 out of 500 females remained as non-maters. Out of the 51 lines, there were 16 lines which produced bisexual offspring. These were discarded, since they were assumed to have mated during the mass screening. Seven of the other 35 lines which consisted of only female offspring were selected randomly and allowed to reproduce parthenogenetically with small mass cultures. Tests of mating speed were carried out using females of the fourth parthenogenetic generation after screenings and males from bisexual strains. Ten replicates for each combination were subjected to homogeneity tests for the number of

8 548 H. IKEDA AND H. L. CARSON females mated within one hour and those remaining unmated. Since all groups were homogeneous, the data were pooled and contingency x2 tests for group comparisons were made on the pooled data. Out of seven lines tested, one line, Sel-l-Im, showed a markedly reduced mating speed both with R (37%) and with S males (50%) (see Table 3). Females of the other six lines mated with rates of 75.6% with R males and 80.8% with S males on the average. The fastest line, Sel-lf-Im, showed mating speed of 83% with R males and 86% with S males. For the original unselected line, RSB-7-Im, 65% of females mated with R males and 82% with S males. The differences in mating speed between Sel-l-Im and RSB-7-Im are highly significant for both R and S males (x2 = and , respectively). Sel-l-Im females mated with 0 males with a rate of 6l%, indicating no significant difference between this line and RSB-7-Im (x2 = ). The lack of the significant difference between them possibly resulted from the fact that a Bridge strain used in the first cycle originated from 0 strain and in addition, that no mass screening was carried out at all with 0 males. Results of the second cycle of selection The result of the mass screening is shown in Table 4, Cycle females each of sl+ and sl were subjected to mass screening separately. During mass screenings, some of them escaped, or were killed accidentally, so the total number of females tested was 254 for sl+ and 258 for sl. After 7 days of mass screenings (3 times with R males, 3 times with S males and once with 0 males), 47 females remained as non-maters. Twenty-two of these were sl+ and 25 females were sl, showing no significant difference between them. Unmated females were placed individually in food vials to establish parthenogenetic lines. Finally 19 sl+ and 18 sl lines were obtained. Others were discarded because either they produced bisexual offspring or no offspring at all. Seven lines each of sl+ and sl were picked up at random and allowed to reproduce parthenogenetically to make stocks large enough to be tested. The tests of mating speed were conducted using females of the third or the fourth generations. Chi-square tests were made in the manner mentioned in a previous paragraph. The average mating speeds were 40.0% with R males and 36.8% with S males for the pooled data of sl+ and sl. These averages are lower than those of the first cycle. The fastest line, Sel-2f-Im, SI+, showed mating speed of 54% with R males and 60% with S males. Sel-2-Im, sl, showed the lowest values of mating speed, 27% with R males and 22% with S males. Although thsese values did not differ significantly from those of several lines, Sel-2-Im was chosen as a representative slow-mating line of the second cycle. Mating speed of Sel-2-Im was 43% with 0 males. The data of Sel-2-Im are shown in Table 3. There are significant differences in mating speed between Sel-l-Im and Sel-2-Im for S males (x2 = ) and 0 males (x2 = ), but not for R males (x2 = ).

9 MATING RELUCTANCE IN D. mercatorum TABLE 5 Isolation indices obtained by the male multiple choice method using bisexual strains of D. mercatorum 549 Female RO : SO SO : 0 9 RO : 0 9 Male Sd -0.14* In all combinations, indices are significantly different from zero (P < 0.001) except for the figure with an asterisk. The degree of sexual isolation The degree of sexual isolation was examined for all possible combinations among the three bisexual strains, and also between bisexual and parthenogenetic strains. Table 5 shows isolation indices for the combinations among the three bisexual strains. All indices except that of (RO 4- SO) X R8, are significantly different from zero (random mating). The matings in which S females were placed together with both sexes either of R or 0 strains gave negative indices, showing that much more heterogamic matings occurred. The results are considered to be due mainly to differences in female receptivity. As shown in Table 2, there is a tendency for S females to mate more often than R and 0 females and to accept all kinds of males with a great intensity. Isolation indices for all possible combinations between the three bisexual strains and the three parthenogenetic strains, including the two selected ones, are given in Table 6. All of the values are significantly different from zero. The original unselected parthenogenetic strain, RSB-7-Im, showed some degree of sexual isolation differently against R, S and 0 strains, showing indices 0.32,0.59 and 0.60, respectively. It is clear however, that sexual isolation between parthenogenetic and bisexual strains was enhanced by selection. The isolation index against R strain doubled after the first cycle of selection. Nevertheless, no further increase was observed in the successive selection cycle. On the other hand, there is no difference in index between RSB-7-Im and Sel-1-Im against 0 males, whereas by the second cycle the degree of sexual isolation was increased. Against the S strain, it was increased in both the first and the second cycles. TABLE 6 Isolation indices obtained for combinations between bisexual and parthenogenetic strains of D. mercatorum Bisexual female and male Impaternate female R S 0 RSB-7-Im Sel-I-Im Sel-ZIm 0.M All indices are significantly different from zero (P < 0.001).

10 550 H. IKEDA AND H. L. CARSON TABLE 7 Fecundity and rate of parthenogenesis of a bisexual and five parthenogenetic strains of D. mercatorum Strain S RSB-7-Im Sel-l-Im Sel-lf-Im Sel-2-Im Sel-2f-Im No. of adults Rate of No. of eggs?? dd parthenogenesis 2, O.M% 4, I ,lM a 7, , , The rate of parthenogenesis There is no direct way to show whether Sel- l-im was produced bisexually or parthenogenetically after the outcross between RSB-7-Im and OB-2-Br in the first cycle. The rate of parthenogenesis was considered to be one of indirect indications to see the difference between RSB-7-Im and Sel-l-Im. If the rate of parthenogenesis is different between two strains, Sel-l-Im would be found to be the bisexually-produced one. On the other hand, to see if the parthenogenetic rate associates with mating speed, the rates were obtained for S, RSB-7-Im, Sel-l-Im, Sel-lf-Im, Sel-2-Im and Sel-2f-Im. Table 7 shows the number of eggs laid, the number of adults emerging and the rate of parthenogenesis. The rate of RSB-7-Im7 4.1%, is similar to 3.2% obtained by CARSON (1967) using the same strain, and is only about one-half of the value of Sel-l-Im, 8.45%. The number of eggs laid by Sel-l-Im is almost twice as great as that of RSB-7-Im. The high egg production of the former seems to be because of the increased heterozygosity caused by outcrossing. From these results, the possibility that Sel-l-Im may be derived parthenogenetically from RSB-7-Im without outcrossing to the Bridge strain can be ruled out. The data presented here also suggest that the parthenogenetic strains formed after outcrossing are different from each other with respect to polygenic characters such as fecundity and parthenogenetic rate. When the parthenogenetic rates of the six strains tested are compared with mating speed, it seems that there is no correlation between the two attributes. DISCUSSION Artificial selection for reluctance to mate on the part of parthenogeneticallyproduced females of D. mercatorum has evidently been successful. A slow-mating line in each cycle was established. The single founder females for these lines were selected out of 500 females which were raised parthenogenetically from F1 offspring of the outcross between a parthenogenetic and a Bridge strain. The response to selection was measured by mating speed shown by the percentages of females mating in a one-hour observation period. Sexual isolation was also measured by STALKER S isolation index (1942). It is noteworthy that the significant differences in mating speed between the original unselected strain and the

11 MATING RELUCTANCE IN D. mercatorum 551 selected one, Sel-l-Im, (against males of two out of three bisexual strains) were observed after the first cycle of selection and were further increased by the second cycle. A second parthenogenetic line, Sel-2-Im, shows an increased reluctance to mate with males of all three bisexual strains. The measures of the mating speed of the parthenogenetic strains might be biassed towards slowness, for the following reason. The parents of the parthenogenetic females remained on food vials for five days in order to obtain the large number of virgin flies necessary. On the other hand, bisexual parental flies were transferred onto fresh food vials every day. Thus, the condition during the larval stage might be different between the two sets of flies. Culture conditions have been found to have effects on mating propensity (SPIESS and SPIESS 1969). The fact, however, that mating speed was promptly reduced by the successive cycles of selection is suggestive that the differences in culture conditions, if any, should not be considered to be a factor large enough to deny the effect of selection. Sexual isolation between the parthenogenetic and the bisexual strains was significantly increased by selection, as measured by the direct observation method employed here. Comparing the isolation index with mating speed, it seems likely that the degree of sexual isolation displayed can be interpreted principally as a difference in mating speed between two strains tested; this is especially true of the mating frequency in Period I, which is the proportion of females mated within the first ten minutes. Thus, the greater the difference in the mating frequency in Period I between homogamic and heterogamic matings, the greater was the isolation index obtained. For example, there is no significant difference in mating speed between the combination of RSB-7-Im and R (61%) and RSB- 7-Im and 0 (62%), yet the isolation index between RSB-7-Im and 0 is about twice as much as that between RSB-7-Im and R. The difference in mating frequency in Period I between homogamic and heterogamic is greater in the former combination than that of the latter; i.e., the mating frequency in Period I of RSB-7-Im females was 70.3% and 50.4% when tested with R and 0 males respectively, whereas the values for homogamic matings were 79.8% for R and 85.9% for 0 strains. Since no selection for male behavior has been conducted at all, it would be concluded that the genes responsible for the sexual isolation observed here seem to be almost entirely associated with female mating behavior, especially with mating speed. In Drosophila, mating speed depends on both courtship intensity of males and female receptivity (MANNING 1961). The possible parts played by the female and the male in mating speed have been described in several Drosophila species. It has been found that mating speed is controlled mainly by the male in D. melanogaster (HOSGOOD and PARSONS 1965). In D. persimilis, on the other hand, the female mainly determines mating speed (SPIESS and LANGER 1964). In D. pseudoobscura, the determination is due to the male in some cases (KAUL and PARSONS 1965; SPIESS, LANGER and SPIES 1966), whereas the female was the determining sex in some other combinations (PARSONS and KAUL 1966). The present study using D. mercatorum shows that the female determination of mating speed is relatively more important than the male influence. When mating

12 552 H. IKEDA AND H. L. CARSON speed of bisexual strains is summarized in reference to the male strain (see RESULTS), it is significantly different for all of the six comparisons. On the other hand, referring to the female strain (Table 2), only three of the six group comparisons show significant differences. Bisexual slow-mating lines were established by artificial selection in D. simu- Zans (MANNING 1968) and also in D. pseudoobscura (KESSLER 1969). In these cases, the females seem to respond to selection for slow mating, whereas the males are not affected by selection for slow mating. KESSLER (1966) estimated the number of genes affecting mating speed to be relatively few in D. pseudoobscura, since the response to selection for mating speed is very rapid, achieving a high value within five generations. Further difference in mating speed between the fast and the slow lines w2s not obtained. HENSLEE (1966) found that there is a significant difference in mating preference between a parthenogenetic strain which was selected for sexual isolation for only one generation and an unselected original strain when tested with wild-type males in D. mercatorum. DOERR (1967) also observed that mating speed was significantly reduced by artificial selection for slow mating in a few generations in a parthenogenetic strain of the same species. From the fact that the degree of reluctance to mate was remarkably increased within a few generations when the Parthenogenetic strain was used, it is likely that the genetic basis of the effect would be simple. Possibly, it involves a few genes rather than polygenic systems, as has been pointed out by KESSLER. The fixation of these genes in the homozygous state may be accelerated by parthenogenetic reproduction. Sexual isolation exists between the unselected parthenogenetic strain, RSB- 7- Im, and bisexual strains. RSB-7-Im was established from a cross between R and S strains in 1964 (CARSON 1967) and had been kept parthenogenetically in the laboratory without selection since its formation. Possibly the founder population of this strain may have had a genetic constitution which favors mating both with R and S males. Brief tests were carried out using the inter-strain F, hybrid females between R and S. These females were found to mate well both with R and S males, giving frequencies of mating within 30 min of 94% with R males and 100% with S males. The genotype which shows some degree of reluctance to mate with males from bisexual strains might have been established by chance relatively rapidly. This is because of recombination and the nature of diploidization in parthenogenesis. This may cause immediate fixation in the homozygous state. However, the possibility that the degree of mating reluctance is correlated with the rate of parthenogenesis can be ruled out, since no correlation between mating speed and the parthenogenetic rate was found (see Table 7). Preliminary studies reported by HENSLEE (1966) and WEI (1968) show that parthenogenetic strains in D. mercatorum show some isolation when tested with normal bisexual males. WASSERMAN (1962) noted that D. mercatorum probably arose originally in the Brazilian and Bolivian lowlands and spread into the Nearctic and Neotropical regions. In addition, this species has been found in the Hawaiian islands and in Australia (PATTERSON and WHEELER 1949), in Spain (PREVOSTI 1950) and in

13 MATING RELUCTANCE IN D. mercatorum 553 Samoa (WHEELER and KAMBYSELLIS 1966). PATTERSON and WHEELER (1949) stated that the introductions could have been made by fruit-carrying boats. WASSERMAN and WILSON (1957) performed intraspecific matings using some geographical strains of D. mercatorum; these proved to be fully interfertile. In the present study, three bisexual strains of this species were used. S strain was derived from the population of La Palma, El Salvador. R and 0 strains were derived from the populations of Rochester, New York and Oahu, Hawaii, respectively. Some degree of sexual isolation was found among these three bisexual strains, although all crosses among them yield fertile offspring. They are clearly different from each other with respect to mating speed (Table 2) and duration of copulation (IKEDA, unpublished). We have noted that the shape of the spermatheca of S females is slightly different from that of R and 0 females, the former being longer than that of the latter. Most of S females were observed to copulate twice or more during a single observation, whereas females of R and 0 strains copulate usually only once ( IKEDA 1971 ). This evidence then suggests that there is significant differentiation in the reproductive system among the three bisexual strains derived from different geographic populations. It is considered likely that as this invasive species spreads, new local populations may be founded by a few migrants moving out from the older populations which have much genetic variability. In such cases, inbreeding could take place. This and/or the effect of genetic drift might be considerable, and might result in the establishment of unique gene pools relative to the reproductive system. HOSGOOD and PARSONS (1967) investigated the differences in mating speed and duration of copulation between strains which were derived from single wild-caught females from the same population in D. mdanogaster and found the genetic heterogeneity in these behavioral characters existed among these strains. There is considerable evidence showing correlation among behavioral, physiological and morphological reproductive characters. It was found that the selected slow-mating female shows an apparent delay in time of receptivity (DOERR 1967; MANNING 1968). Possible relationship between the concentration of juvenile hormone and female receptivity has been observed by MANNING (1967). STALKER (1956) suggested that a shortage of males in a bisexual species might provide an opportunity for the evolution of a diploid parthenogenetic biotype beginning from low rates of facultative parthenogenesis. If such shortages were periodically alleviated and bisexual reproduction temporarily resumed, polygenic variance for the trait could be maintained. If this occurs, further selective advance in the efficiency of parthenogenesis could be accomplished even in a species which has a strong tendency toward homozygosis following parthenogenesis. The present experiments add a new dimension to the theory of the origin of diploid parthenogenetic biotypes. They demonstrate in D. mercdorum that genetic variance is present which will permit selection for pertinent behavioral characteristics. These are of such a nature that they tend to isolate the diploid parthenogenetic biotype from sexual reproduction. Thus, following only two cycles of selection, considerable advance in female reluctance to mate was observed. Such isolation, however, is still a long way from what would be neces-

14 554 H. IKEDA AND H. L. CARSON sary to permit coexistence of bisexual and parthenogenetic forms, as in Solenobia (SEILER 1961). Although the same is also true of efficiency of parthenogenesis, the heritability of both the reproductive and behavioral attributes provides evidence that such a mode of evolution of parthenogenetic biotypes from bisexual ancestors is possible. The mode of diploidization in parthenogenetic strains of D. mercatoirum results in about 90% of the impaternate progeny being homozygous (CARSON, WEI and NIEDERCORN 1969). When this system is alternated with outcrossing, as was done by us, a particularly powerful field of variability is provided on which selection can operate. Thus, following an outcross, unmated females generate progeny which consists of a great number of recombinant genotypes fixed in the homozygous state. Thus, outcrossing is automatically followed by a system of extreme inbreeding. Some of the derivatives from such a series of events may by chance fix the genetic basis of behavioral traits which, in this case at least, may lead to isolation of the deme, at least in part, from sexual reproduction. The authors wish to express their cordial thanks to PROFE~SOR H. D. STALKER, Washington University at St. Louis, for his suggestions and criticisms, and also to DR. 0. KITAGAWA for careful reading of the manuscript. This study was supported by a grant GB 7754 from the National Science Foundation and Public Health Service Grant 5504 FR LITERATURE CITED CARSON, H. L., 1967 Selection for parthenogenesis in Drosophila mercatorum. Genetics 55: , 1973 The genetic system in parthenogenetic strains of Drosophila mercaforum. Proc. Nat. Acad. Sci. U.S. 70: CARSON, H. L. and S. H. SNYDER, 1972 Screening for induced mutations by parthenogenesis in Drosophila mercatorum. Egyp. Jour. Genet. Cytology 1 : CARSON, H. L., I. Y. WEI and J. A. NIEDERCORN, JR., 1969 Isogenicity in parthenogenetic strains of Drosophila mercatorum. Genetics 63 : DOERR, C. A., 1967 Artificial selection for sexual isolation within a species. Master s thesis, Washington Univ., MO. HENSLEE, E. D., 1966 Sexual isolation in a parthenogenetic strain of Drosophila mercatorum. Am. Naturalist 100: HOSGOOD, S. M. W. and P. A. PARSONS, 1965 Mating speed difference between Australian populations of Drosophila melanogaster. Genetica 36: , 1967 Genetic heterogeneity among the founders of laboratory populations of Drosophila melanogaster. Austr. Jour. Biol. Sci. 20: IKEDA, H., 1971 Sexual behavior in Drosophila. I. Multiple copulation in Drosophila mercatorum. (Abstr. in Japanese). Jap. Jour. Genetics 46: , 1972 Sexual behavior in Drosophila. 11. Mating competition between two strains of Drosophila mercatorum. (Abstr. in Japanese). Jap. Jour. Genetics 47: 348. KAUL, D. and P. A. PARSONS, 1965 The genotypic control of mating speed and duration of copulation in Drosophila pseudoobscura. Heredity 20 : KFSSLER, S., 1966 Selection for and against ethological isolation between Drosophila pseudoobscura and Drosophila persimilis. Evolution 20: , 1969 The genetics of Drosophila mating behavior. 11. The genetic architecture of mating speed in D. pseudoobscura. Genetics 62:

15 MATING RELUCTANCE IN D. mercatorum 555 MANNING, A., 1961 The effects of artificial selection for mating speed in DrosophiZa melanogaster. Anim. Behav. 15: , 1967 The control of sexual receptivity in female Drosophila. Anim. Behav. 15: , 1968 The effects of artificial selection for slow mating in Drosophila simulans. I. The behavioral changes. Anim. Behav. 16: PARSONS, P. A. and D. KAUL, 1966 Mating speed and duration of copulation in Drosophila pseudoobscura. Heredity 21: PATTERSON, J. T. and M. R. WHEELER, 1949 Catalogue of described species belonging to the genus Drosophila, with observations on their geographical distribution. Univ. Texas Publ. 4920: PREVOSTI, A., 1950 Two newly introduced species of Drosophila found in Europe. Drosophila Inform. Sew. 27: 110. SEILER, J., 1961 Untersuchungen iiber die Entstehung der Parthenogenese bei SoZenobia triquetrella F. R. (Lepidoptera, Psychidae) 111. Zeitsch. f. Vererbungslehre 92: , 1966 Die Zytologie der Bastarde aus der Rreuzung Parthenogenetischer Weibchen von Solenobia triquetrella F. R. mit Mannchen der Bisexuellen Rasse I. Mitteilung. Chromosoma 19: a , Zytologie und Morphologie der Bastarde aus der Kreuzung der diploid parthenogenetischen 2nQ x a. Molec. Gen. Genetics lv2: SPIESS, E. B. and B. LANGER, 1964 Mating speed control by gene arrangement carriers in Drosophila persimilis. Evolution 18 : sE. SPIESS, E. B., B. LANGER and L. D. SPIESS, 1966 Mating control by gene arrangements in Drosophila pseudoobscura. Genetics 54: SPIESS, E. B. and L. D. SPIES, 1969 Mating propensity, chromosomal polymorphism, and dependent cmditions in Drosophila persimilis. 11. Factors between larvae and between adults. Evolutim 23 : STALKER, H. D., 1942 Sexual isolation studies in the species complex Drosophila virilis. Genetics 27: , 1956 On the evolution of parthenogenesis in Lonchoptera (Diptera). Evolution 10 : WASSERMAN, M., 1960 Cytological and phylogenetic relationships in the repleta group of the genus Drosophila. Proc. Nat. Acad. Sci. U.S. 46: , 1962 Cytological studies of the repleta group of the genus Drosophila, 111. The mercatorum subgroup. Univ. Texas Publ. 6205: WASSERMAN, M. and F. D. WILSON, 1957 Further studies on the repleta group. Univ. Texas Publ. 5721: WEI, I. Y., 1968 Mode of inheritance and sexual behavior in the parthenogenetic strains of Drosophila mercatorum. Master s thesis, Washington Univ., MO. WHEELER, M. R. and M. P. KAMBYSELLIS, 1966 Notes on the Drosophilidae (Diptera) of Samoa. Univ. Texas Publ. 6615: Corresponding editor: T. PROUT