SEARCH

SEARCH BY CITATION

Keywords:

  • Drosophila melanogaster;
  • fecundity;
  • fertility;
  • insecticide resistance;
  • longevity;
  • postzygotic isolation;
  • prezygotic isolation;
  • speciation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

Abstract The study of the early stages of speciation can benefit from examination of differences between populations of known history that have been separated for a short time, such as a few thousands of generations. We asked whether two lines of Drosophila melanogaster that were isolated more than 40 years ago have evolved differences in life-history characters, or have begun to evolve behavioural or postzygotic isolation. One line, which is resistant to DDT, showed lower egg production and a shorter lifespan than a susceptible line. These differences are not a pleiotropic effect of resistance because they are not attributable to the chromosome that contains the resistance factors. The two lines have begun to become behaviourally isolated. Again, the isolation is not attributable to genes on the chromosome that contains resistance factors. The lines show only prezygotic isolation; there is no evidence of reduced fitness of F1 or F2 hybrids. These lines and others like them, should be excellent subjects for analyses of genetic changes that could lead to speciation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

The study of speciation can benefit from examination of differences between populations that have been separated for varying lengths of time, from comparisons of species (e.g. Coyne, 1996; Boake et al., 1997) to comparisons of geographically separated populations (e.g. Markow, 1991; Wu et al., 1995). On a shorter time scale, old laboratory lines can be seen as representing the early stages of allopatric divergence, sometimes being ecologically distinct (if they were subjected to different selection pressures), and sometimes ecologically similar, although having had the opportunity to change as a result of factors like genetic drift, mutation and recombination. Ideally, these three levels of increasing time since isolation – laboratory populations, geographical populations and species – could provide cross-sections of the process of speciation. The two levels representing longer isolation have been studied in the genetically tractable organism, Drosophila melanogaster. Here we describe a comparison of two laboratory lines of D. melanogaster that have been separated for somewhat more than 40 years; one was selected for DDT resistance in 1955–56 and the other is an unselected control line (Merrell & Underhill, 1956). We asked whether the stocks had begun to develop behavioural isolation or had diverged in life-history characters.

If stocks that were selected for divergence in the past have experienced relaxed selection for many generations, how do they change? For some textbook cases such as bristle number in D. melanogaster, the selected lines revert towards the value in the base population (Falconer & Mackay, 1996). In one case, cannibalism in Tribolium beetles, differences between lines persisted for at least 60 generations after selection ended (Stevens, 1994). We describe studies of a DDT-resistant line that has maintained its resistance for many decades despite relaxed selection for resistance (Sundseth et al., 1989; Waters et al., 1992a,b). Both the resistant and the control lines showed differences in life-history traits in the mid-1960s, about a decade after selection for resistance was begun (Underhill & Merrell, 1966). We asked whether these differences have persisted.

In 1952, a freshly caught stock of D. melanogaster was divided into two lines. One line was selected for DDT resistance by exposing adults to paper that had been saturated with DDT (called 91R; Merrell & Underhill, 1956). In the early generations, the dose of DDT was low, and it was increased in subsequent generations; a control line, 91C, was not exposed to DDT. In the mid-1960s, the lines showed differences in life histories, as described below (Underhill & Merrell, 1966). Selection continued into the mid-1970s (Dapkus & Merrell, 1977). Line 91R is fixed for the genetic factor(s) that confer DDT resistance and is the focus of investigations of the genetic basis of this resistance (Dapkus & Merrell, 1977; Dapkus, 1992; Maitra et al., 2000). These two lines have been isolated from each other and from other stocks for more than 40 years, or about 1100 generations per line, based on a 2-week generation time.

We re-evaluated the stocks for life-history differences, following as nearly as possible the protocols used by Underhill & Merrill (1966), who proposed that the differences might disappear over time. Further, we tested the lines for behavioural isolation, comparing them to each other and to a common laboratory stock, Oregon-R, which was established in the 1920s (Ashburner, 1989). The factors that confer resistance in 91R have been mapped to two different loci on chromosome II (Houpt et al., 1988; Waters & Nix, 1988; Dapkus, 1992). Resistance has also been associated with over-expression of certain cytochrome P450 genes on chromosome II (Waters et al., 1992a,b; Maitra et al., 1996, 2000; Dombrowski et al., 1998; Maitra, 2000) which are negatively regulated by loci on chromosome III in wild-type strains (Maitra et al., 2000). We used reciprocal chromosomal substitution lines for chromosome II to test whether the life-history traits that differ between the stocks are detectably influenced by genes on this chromosome, which would suggest a close association between DDT resistance and the life-history traits.

We hypothesized that if there is a trade off between resistance and life histories, 91R would show a reduction in one or more of the life-history traits of fecundity, fertility and lifespan. Similarly, we predicted that some degree of behavioural isolation would exist between 91R and 91C, consistent with the reports of other selected lines (Rice & Hostert, 1993). Further, we examined the fecundity and fertility of F1 and F2 hybrids between the lines; these observations allowed us to examine the possibility of post-mating isolation. We found reduced fecundity and longevity but not fertility in 91R, and we also found significant behavioural isolation from 91C. We found no evidence for postzygotic isolation. These effects do not appear to be direct consequences of genes on the chromosome that contains the DDT resistance factor(s). We present these data as an example of the way in which laboratory lines of D. melanogaster can be used to study the early stages of speciation, in the hope of stimulating further investigations on such lines.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

We reared the flies with standard methods, using plastic Drosophila rearing bottles kept in environmental chambers maintained at 24 °C with a 12-h light/dark cycle. The food was an agar-cornmeal-molasses medium to which brewers' yeast was added. Our stocks were maintained in bottles and vials with live yeast added; the ‘no yeast’ condition to which we refer below refers to the absence of live yeast. For the tests of life history components, we used the stocks 91R, 91C and Oregon-R. For some tests of reproductive isolation we used reciprocal chromosome II and III substitution lines (Maitra, 2000) that were developed from 91R and 91C in addition to the three inbred stocks. The chromosome substitution stocks (CRC and RCR) were synthesized by using following second chromosome balancer stocks: Sp/SM5 al2Cy1sp2 and Sco/CyO, Cy; kar ry506; and the third chromosome balancer stock TM3 Sb ry e/TM6 Tb e. The first was obtained from the Bowling Green Mid America Stock Center and the other two were obtained from Dr John Lucchesi (Emory University). The details of the stocks are described in Lindsley & Zimm (1992). Statistical analyses were conducted with JMP (SAS Inc., Cary, NC, USA).

Life-history traits

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

We evaluated the life history components of fecundity, fertility and longevity, using methods that were as similar as possible to those reported by Underhill & Merrell (1966); these data were collected in 1999–2000.

For the comparisons of fecundity and fertility, we collected data from individual females. At the start we housed a single virgin female and three males together in an unyeasted food vial. We transferred these flies to a new vial every day and counted eggs in the previous vial immediately after transfer. The vials were frozen when the flies had emerged, 10–12 days after the eggs were counted, the frozen adults were counted. We collected data from at least 18 females of each stock. Fecundity was computed from total egg production over the first 20 days of each female's life, and expressed as the average number of eggs per female per day. Initially we had collected eggs for each female's entire life, but after measuring the first block of females we found that egg production for the first 20 days was highly correlated with lifetime egg production (r2=0.67, P < 0.0001). This result is probably because about 66% of the lifetime production occurs in the first 20 days despite some flies living nearly 80 days.

We expressed fertility as the ratio of the number of adult offspring per female to the number of eggs laid, summing over the 20 days. Underhill & Merrell (1966) reported percent fertility, but based their values on estimates of lifetime egg production because they only recorded egg production for alternate 3-day intervals. Our data reflect counts made on every day of the study.

We measured longevity by placing a group of 50 virgins, 1-day-old flies into an unyeasted food vial for 24 h. Each day the flies were transferred to a new vial and the dead flies counted, until all the flies were dead. We recorded the median survival in each vial. These methods copy those of Underhill & Merrell (1966) except that they used bottles and changed them weekly. We tested two blocks of five vials each for each stock, collecting data for each stock at the same time. We used rank-sum tests to compare life-history measures between genotypes.

Fecundity and fertility of hybrids

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

We carried out a second study of life histories to test whether inbreeding caused the differences between 91R and 91C in fecundity and fertility. The production of F1 progeny gives information about prezygotic (but post-mating) isolation and postzygotic isolation. For example, if fewer adults were to emerge than the number of eggs that were in the vial, the reason could be the failure of fertilization (prezygotic isolation) or failure of development (postzygotic isolation). The production of F2 progeny allowed us to assess the fecundity and fertility of F1 hybrids, two additional measures of postzygotic isolation. Our methods were similar to our earlier methods except that the virgin females were 2–7 days old at the start of the experiment, were housed with one to three males (generally two), and eggs were collected for only 5 days. As with the first experiment, vials were unyeasted. A total of 20 females per genotype was tested. These tests were distributed over 5–10 blocks for every genotype; 91R and 91C provided two or three replicates in every block in order to control for environmental effects. We tested the parental lines, reciprocal F1 hybrids and F2 hybrids generated from crossing within the reciprocal F1 lines. We also tested the reciprocal chromosome II substitution lines, RCR and CRC. Fecundity was defined as the total eggs per female for 5 days, and fertility was the ratio of number of adults plus uneclosed pupae to number of eggs, adjusted to 1.0 if we counted more adults than eggs (sometimes eggs were piled on each other and hard to count). Uneclosed pupae probably represent differences in developmental time (R. Ganguly, pers. obs.) rather than pupal death; in the few cases that we held a vial with uneclosed pupae for an additional day or two, they all emerged.

Groups were compared with nonparametric tests. The hypotheses to be tested (e.g. that the reciprocal F1 progeny did not differ) did not apply to the entire data set, and thus most comparisons reported below are based on pairwise tests. We used rank-sum tests. Because related data were used in many tests, we used a sequential Bonferroni procedure to evaluate the significance of the comparisons made with these stocks at alpha=0.05.

Tests of behavioural isolation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

We used multiple-choice tests to examine the possibility of prezygotic isolation between the stocks. For each experiment, 40 virgin flies of each sex were sorted within 8 h of eclosion. They were held for 5–10 days before being tested. The day before the behavioural tests, they were transferred to Carolina instant Drosophila medium that had been dyed with red or green food colouring. We housed flies on colours corresponding with genotype, and alternated the colours for each test. A statistical analysis of our behavioural results showed that neither colour resulted in net assortative mating.

For each test, we released 160 flies (40 of each sex and genotype) into a Plexiglas chamber (28 × 22 × 22 cm). We observed the group of flies for 20 min, or until 20 pairs had mated, whichever came first. During the test we aspirated mating pairs from the chamber and froze them. The genotypes of the flies were scored by the colour of their abdomens. We conducted tests until we had collected data for about 100 pairs per comparison, or at least five tests per comparison. By scoring only 20 pairs in each group of 160 flies, we hoped to keep the sampling variances approximately the same during the test, so that all individuals had a high probability of encountering flies of the other genotype (Casares et al., 1998 recommend that no more than the first 50% of mating pairs be scored, so our methods were conservative). Each fly was tested just once. We tested for assortative mating by using χ2 contingency tables. These tables can also be used to evaluate mating propensity by examining the marginal totals for each sex of each line.

Life-history traits

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

In overall fecundity, 91C was significantly higher than both Oregon-R and 91R in 1999–2000. The 91R had significantly lower numbers of adults and longevity than the other stocks (Table 1). We present the data from Underhill & Merrell (1966) for comparison; as noted above, our methods differed from theirs in ways that do not allow us to compare the numbers directly. Fecundities for all stocks were far lower in 1999–2000 than in 1966; we cannot test whether this was the result of rearing methods or because of long-term changes in the stocks. Rank orders differed between the studies. In 1966, the fecundity of 91C was close to that of the other stocks but in 1999–2000 it had the highest fecundity. The rank orders for fertility differ between studies but in both studies, 91R had the lowest fertility. In 1966, 91R had the greatest longevity, but in 1999–2000 it had the shortest median adult lifespan. Thus 91R appears to show more of a fitness decrement in 1999–2000 than it did in 1966.

Table 1.  Life-history measures for three lines, comparing data from 1966 to 1999–2000.
GenotypeFecundityFertilityMedian longevity, days,
1999–200019661999–200019661999–20001966
  1. Fecundity is expressed as the number of eggs per female per day. For the 1999–2000 data on fecundity and fertility, mean ± 1 SE (18 replicates each for 91R and Ore-R, 21 for 91C); for longevity, median and range. The 1966 data are modified from tables in Underhill & Merrell (1966), where fecundity is the average lifetime egg production per female. Stocks differed significantly in 1999–2000 for fecundity, fertility and longevity (P=0.006, 0.005 and 0.001, respectively, rank-sum tests).

91R7.4 ± 0.630.9 ± 0.90.73 ± 0.030.6439 (35–42)48.5
91C10.1 ± 0.931.8 ± 1.20.88 ± 0.020.7252 (34–58)42.5
Oregon-R6.1 ± 0.730.4 ± 1.40.80 ± 0.040.8654 (42–62)47.5

Tests of isolation: fecundity and fertility of hybrids

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

Here we also found that 91R females produced significantly fewer eggs than 91C (Fig. 1; P < 0.05, Bonferroni adjusted). The fitness (production of adults) of 91R relative to 91C was 71%; the F1 and F2 fitness were 81 and 90% of 91C, respectively. The ratio of adults to eggs produced did not differ significantly between 91C and 91R. Thus the difference in the numbers of adults resulted from egg production rather than larval or pupal survival. Our 1999–2000 data (Table 1) showed lower egg-to-adult survival for 91R than 91C; the difference could be a result of different persons handling the flies in each study, or the result of differences in environmental conditions such as food or humidity.

image

Figure 1. Average number of eggs (top), number of adults (centre), and survival (bottom) for the selected lines and their crosses; error bars are 2 SE. Each value is the mean of data from 20 females.

Download figure to PowerPoint

The reciprocal F1 hybrids did not differ from each other for any trait measured (P > 0.05), so the data were combined. Postzygotic isolation would be indicated if some of these traits had lower values in the hybrid than in the parental generations, but this pattern is not seen. The mean values were more similar to 91R than to 91C (Fig. 1), suggesting some dominance of the DDT-resistant genotype for egg production. The reciprocal F2 hybrids were also sufficiently similar to allow combining the data (P > 0.05). Their mean values were intermediate between those of the two parental lines (Fig. 1). For both the F1 and F2 generations, differences in numbers of adults are attributable to the numbers of eggs produced rather than to differential survival. The F1 and F2 lines appear to have slightly higher values than the parental lines for egg-to-adult survival, a more comprehensive measure of fitness (Fig. 1c). The similarities of F1 and F2 progeny to the parental lines in these measures means that postzygotic isolation is undetectable.

One of the two chromosomal substitution lines, RCR, was significantly lower than the other (CRC) for the number of adults produced (P < 0.05, Bonferroni adjusted), but the difference in the number of eggs was not significant when adjusted for multiple comparisons within the data set. RCR had significantly lower egg-to-adult survival than CRC (P < 0.05, Bonferroni adjusted); thus the reduced number of adults produced by RCR is largely an effect of reduced egg-to-adult survival. RCR did not differ significantly from 91R for any trait except egg-to-adult survival but it differed significantly from 91C in both egg production and number of adults (P < 0.05, Bonferroni adjusted).

Tests of isolation: mating behaviour

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

We found significant behavioural isolation between 91R and 91C, but not between either line and Oregon-R (Table 2). Despite the significant difference, the homotypic mating frequency of 91R with 91C was modest. The difference between 91R and 91C is not the result of different mating propensities, because similar numbers of each line and each sex mated. In contrast, Oregon-R females had a substantially higher mating propensity than 91R females and a lower mating propensity than 91C females, in tests with each line.

Table 2.  Tests of assortative mating between lines.
Line ALine BNumber of matingsTotal matingsChi-square valueProportion matings homotypic
A × AB × BA × B*B × A*
  • *

    Genotype of female is given first.

  • P  < 0.05 (Bonferroni adjustment for multiple comparisons).

  • Results in each comparison are the sum of matings from at least five independent 20-min multiple-choice tests with 160 flies per test, and no more than 20 pairs scored per test.

Tests between stocks
 91R91C3035152310316.850.63
 91ROregon-R174113351060.740.55
 91COregon-R411624211020.180.56
Tests with chromosomal substitution lines
 91RCRC283829181133.9960.58
 91RRCR202414339100.48
 91CCRC31191034941.820.53
 91CRCR3949144014212.180.62
 CRCRCR19341926980.690.54
 CCRRRC50131520983.630.64

If behavioural isolation is a pleiotropic consequence of DDT resistance, then either chromosome II or III could play a role in isolation, because both chromosomes contain factors that contribute to resistance (Dapkus & Merrell, 1977; Dapkus, 1992). We focused on chromosome II because the factors on it that are associated with resistance appear to be over-expressed (Waters et al., 1992a,b; Maitra et al., 1996, 2000; Dombrowski et al., 1998). We hypothesized that if chromosome II is involved in isolation, comparisons containing its substitution (CRC × 91R and RCR × 91C) should show random mating, because both genotypes within a comparison would bear a second chromosome of the same origin. Similarly, comparisons of lines with nonsimilar substitutions (CRC × 91C and RCR × 91R) should show nonrandom, homotypic matings, because the two lines being compared would have different second chromosomes. In contrast to these predictions, we found significant homotypic mating only for 91C × RCR (and not quite significant for 91R × CRC), that is, when both lines tested bore a second chromosome of the same genotype (Table 2). Further, the tests between lines bearing different second chromosomes but similar X and third chromosomes showed random mating (91C × CRC, and 91R × RCR). The test of CRC with RCR showed random mating whereas the test of 91C with RCR showed strong isolation; the difference between the two tests is the identity of the second chromosome in one stock (R in CRC, C in 91C), which indicates that the second chromosome could have an effect, perhaps in interaction with one or more other chromosomes. The test between the lines bearing reciprocal substitutions of chromosome III (CCR, RRC) showed a nearly significant nonrandom mating, but this may be the result of the far greater mating propensity of both males and females of CCR.

Life history and DDT resistance

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

The lines 91R and 91C can be considered to have diverged in allopatry after experiencing an initial ecological difference, exposure to DDT. Underhill & Merrell (1966) reported on several pairs of resistant and control lines; they found that resistant flies had greater longevity in all comparisons. Young adults from their control populations tended to have greater fecundity and fertility. They found very high lifetime fecundity of 91R (their Table IV), which they explained as a result of the longer lives of flies in the resistant lines. The authors suggested that longevity would have been favoured in their selected lines because the strip of paper that contained DDT gradually was covered with debris, and therefore longer-lived flies could lay eggs that were exposed to lower doses of DDT. Thus the selection regime favoured late-life egg production, and longer lives, which could produce the greater lifetime productivity that the authors saw in resistant strains. We note that although we report comparisons with lines called Oregon-R from both our study and the 1966 study, we are not under the illusion that the name applies to the same line (Ashburner, 1989). The Oregon-R with which Underhill & Merrell (1966) worked was a propagule of Oregon-R that had been inbred for 25 generations at the start of their experiment. Oregon-R is generally considered to represent a wild type (that is, it bears no obvious visible mutants); we used it for the sake of comparison, in an attempt to decide if one or both inbred lines show reduced fitness.

Our research was conducted after many generations of relaxed selection for DDT resistance (perhaps 20 years), which have nevertheless not resulted in the loss of resistance. For each of the life-history traits measured in 1999–2000 and for fecundity measured in 2001, the resistant line had a lower value than the control line (Table 2, Fig. 1), which may indicate some cost of resistance. Thus since the mid-1960s, 91R appears to have lost its increased longevity but maintained decreased fecundity and fertility. These long-term changes may indicate a cost of resistance that is too weak to detect in short-term studies. Life-history costs of insecticide resistance have been reported in some but not all studies (Carrière & Roff, 1995; Chevillon et al., 1997; Shufran et al., 1997). Another hypothesis to explain the decreased fitness of 91R, inbreeding depression, seems unlikely because neither the F1 or F2 progeny of crosses with 91C showed increased fitness. Our results would be consistent with inbreeding depression only in the unlikely circumstance of both 91R and 91C being fixed for the same alleles that tend to decrease fitness. That is, if fitness is a polygenic trait, then decreases in fitness in different lines are most likely to be the result of fixation at different loci, in which case the F1 hybrids should show increased fitness as a result of complementation. Thus we are left with the problem of why resistance has been maintained despite relaxed selection for resistance and some life-history cost. The answer may be that 91R was maintained in laboratory culture regardless of its productivity, and that active intervention allowed it to persist.

Lines that were selected for divergence could show divergence in unselected traits because of pleiotropic effects of the genes influencing the targets of selection, or because of drift during the selection process. If a trade off between life-history characters and resistance was because of pleiotropy, the spread of insecticide resistance in natural populations could become slower (Carrière & Roff, 1995). In our tests, if reduced fitness is a pleiotropic effect of alleles carried by genes that confer DDT resistance, then CRC should show reduced fitness compared with 91C. In contrast, we found reduced fitness in RCR compared with the parental lines and CRC. This might be explained if the R-type chromosome III produced over-expression of genes on the C-type chromosome II in RCR individuals, and this over-expression had a cost. An explanation of the difference between RCR and 91R would require the additional hypothesis that 91R had evolved mechanisms to compensate for the costs of over-expression.

Evolution of behavioural isolation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

Behavioural isolation has begun to evolve between 91R and 91C. However, neither line is isolated from Oregon-R (Table 2). This result could be because of sample size, although nearly 400 tests would be needed to detect isolation between either line and Oregon-R with the 55% homotypic mating that we measured. Our result would be explained if 91R and 91C had moved in different directions from a midpoint expressed in Oregon-R. Other stocks of D. melanogaster that were divergently selected in allopatry for traits such as positive and negative geotaxis have been tested for behavioural isolation. In 10 of the 14 such studies reviewed by Rice & Hostert (1993), significant behavioural isolation was detected in at least some comparisons, even after relatively few generations of divergence. Thus our behavioural results are not surprising.

To our knowledge, laboratory stocks that have evolved some level of behavioural isolation have rarely if ever been exploited for studies of the genetic basis of behavioural divergence. When we tested whether chromosome II had a strong influence on isolation between the two lines, we found the opposite, that lines that shared the second chromosome were isolated (Table 2). This suggests that the X chromosome or chromosome III, or both, may contribute substantially to the isolation between 91R and 91C. The results of the test of CRC with RCR suggest that an interaction between chromosomes could also influence isolation. The tests of flies from the chromosome III substitution lines were not informative because of the far higher mating propensity of both males and females from CCR. We did not have lines available to allow tests of the influence of the sex chromosomes. If chromosome III is important in behavioural isolation, then tests of CCR and RRC with 91R and 91C should show homotypic mating when the third chromosomes differ.

Our tests of behavioural isolation were all multiple-choice tests. When populations are in the early stages of divergence, different types of tests (usually called multiple-choice, choice by one sex, and no-choice) may not show the same magnitude of behavioural isolation (Markow, 1981; Fraser & Boake, 1997). We have begun to observe single pairs; these suggest that there are slight differences in courtship between 91R and 91C that might account for the isolation seen in multiple-choice tests. Further observations of single pairs are planned as a preliminary step to the genetic analysis of behavioural isolation between these lines.

Evolution of postzygotic isolation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

Our analyses of fecundity and fertility of F1 and F2 hybrids indicate that no detectable postzygotic isolation has begun to evolve. In previous studies of postzygotic isolation, a stringent criterion of sterility or inviability of at least one sex of the hybrids was used (Coyne & Orr, 1989, 1997). The more laborious tests that we conducted should allow the detection of incipient post-mating isolation, which could then be examined further to learn whether it represents problems with fertilization (e.g. Alipaz et al., 2001) or postzygotic isolation. Ringo et al. (1985) found some evidence of post-mating isolation after divergent selection for several traits in D. simulans (based on a re-analysis by Rice & Hostert, 1993). It might be preferable to modify our methods by pairing each F1 female with a single male to study F2s, because both empirical and theoretical studies indicate that male Drosophila are likely to evolve postzygotic isolation before females (Orr, 2001).

Rice & Hostert (1993 ) reviewed data on laboratory studies of population isolation; they found evidence for evolution of prezygotic but not postzygotic isolation. However, the longest reported opportunity for allopatric divergence was 5 years ( Kilias et al., 1982 ). Further, Kilias et al . (1982) reported decreases in fitness of some crosses in their 5-year study which suggests that postzygotic isolation might begin to evolve in laboratory stocks. The absence of postzygotic isolation in our data is consistent with Rice & Hostert's (1993 ) conclusions.

Models of speciation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

Coyne & Orr (1989, 1997 ) proposed that sympatric divergence should result in more rapid evolution of prezygotic isolation than allopatric divergence, which they attributed to reinforcement. In their reviews of prezygotic and postzygotic isolation in the genus Drosophila , they found that the two kinds of isolation evolved at approximately the same rate in allopatric taxa ( Coyne & Orr, 1989, 1997 ). Our data on isolation between 91R and 91C would give a value of 0.42 for prezygotic isolation [1 – (prop heterotypic matings/prop homotypic matings)]; and 0 for postzygotic isolation, but Coyne & Orr (1989 ) found greater prezygotic than postzygotic isolation for only two of eleven comparisons of allopatric species. Thus laboratory selection can result in extensive isolation. Further studies of fly populations that have the number of years of separation that we examined might shed light on the stages of speciation.

Recent population genetic models of the process of speciation indicate that both genetic architecture and mating behaviour can have strong influences on the probability and rate of speciation (e.g. Lande, 1981; Turner & Burrows, 1995; Gavrilets & Hastings, 1996; Gavrilets & Boake, 1998). Despite these inducements to investigate the genetics of behavioural isolation, progress in the field has been slow (Howard & Berlocher, 1998). Some scientists investigate the genetic architecture of behavioural differences in species that diverged long ago (e.g. Coyne, 1996; Shaw, 1996); such studies are valuable for allowing us to understand the maintenance of species boundaries. However, in order to understand the genetic changes that accompany the initiation of speciation, nascent species need to be examined. One way to do this is to find diverging natural populations, as has been performed very successfully with African populations of D. melanogaster (Hollocher et al., 1997; Ting et al., 2001) and cactophilic Drosophila (Markow, 1991; Etges, 1992). Our approach is to investigate laboratory stocks that were isolated decades ago. These stocks have maintained life-history differences despite relaxed selection, and have developed statistically significant behavioural isolation. We believe that such stocks could be very powerful tools for studying the divergence that could proceed to speciation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References

We thank Massimo Pigliucci and two anonymous reviewers for valuable comments on the manuscript. We are grateful to the undergraduates who provided help with stock keeping. This research was assisted by funding provided by the US National Science Foundation (grant IBN-9514041 to C.B) and the US Department of Agriculture (grant 99-35302-8081 to R.G).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Life-history traits
  6. Fecundity and fertility of hybrids
  7. Tests of behavioural isolation
  8. Results
  9. Life-history traits
  10. Tests of isolation: fecundity and fertility of hybrids
  11. Tests of isolation: mating behaviour
  12. Discussion
  13. Life history and DDT resistance
  14. Evolution of behavioural isolation
  15. Evolution of postzygotic isolation
  16. Models of speciation
  17. Acknowledgments
  18. References
  • Alipaz, J.A., Wu, C.-I. & Karr, T.L. 2001. Gametic incompatibilities between races of Drosophila melanogaster. Proc. R. Soc. Lond. B. 268: 789795.
  • Ashburner, M. 1989. Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
  • Boake, C.R.B., DeAngelis, M.P. & Andreadis, D.K. 1997. Is sexual selection and species recognition a continuum? Mating behavior of the stalk-eyed fly Drosophila heteroneura. Proc. Natl. Acad. Sci. USA 94: 12 44212 445.
  • Carrière, Y. & Roff, D.A. 1995. Change in genetic architecture resulting from the evolution of insecticide resistance: a theoretical and empirical analysis. Heredity 75: 618629.
  • Casares, P., Carracedo, M.C., Del Rio, B., Piñeiro, R., Garcia-Florez, L. & Barros, A.R. 1998. Disentangling the effects of mating propensity and mating choice in Drosophila. Evolution 52: 126133.
  • Chevillon, C., Bourguet, D.F.R., Pasteur, N. & Raymond, M. 1997. Pleiotropy of adaptive changes in populations: comparisons among insecticide resistance genes in Culex pipiens. Genet. Res., Camb. 70: 195204.
  • Coyne, J.A. 1996. Genetics of sexual isolation in male hybrids of Drosophila simulans and D. mauritiana. Genet. Res., Camb. 68: 211220.
  • Coyne, J.A. & Orr, H.A. 1989. Patterns of speciation in Drosophila. Evolution 43: 362381.
  • Coyne, J.A. & Orr, H.A. 1997. ‘Patterns of speciation in Drosophila’ revisited. Evolution 51: 295303.
  • Dapkus, D. 1992. Genetic localization of DDT resistance in Drosophila melanogaster (Diptera: Drosophilidae). J. Econ. Entomol. 85: 340347.
  • Dapkus, D. & Merrell, D.J. 1977. Chromosomal analysis of DDT-resistance in a long-term selected population of Drosophila melanogaster. Genetics 87: 685697.
  • Dombrowski, S.M., Krishnan, R., Witte, M., Maitra, S., Diesing, C., Waters, L.C. & Ganguly, R. 1998. Constitutive and barbital-induced expression of the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic background of a low producer strain. Gene 221: 6977.
  • Etges, W.J. 1992. Premating isolation is determined by larval substrates in cactophilic Drosophila mojavensis. Evolution 46: 19451950.
  • Falconer, D.S. & Mackay, T.F.C. 1996. Introduction to Quantitative Genetics, 4th edn. Longman, New York, USA.
  • Fraser, I. & Boake, C.R.B. 1997. Behavioral isolation, test designs, and Kaneshiro's hypothesis. Am. Nat. 149: 527539.
  • Gavrilets, S. & Boake, C.R.B. 1998. On the evolution of premating isolation after a founder event. Am. Nat. 152: 706716.
  • Gavrilets, S. & Hastings, A. 1996. Founder effect speciation: a theoretical reassessment. Am. Nat. 147: 466491.
  • Hollocher, H., Ting, C.-T., Pollack, F. & Wu, C.-I. 1997. Incipient speciation by sexual isolation in Drosophila melanogaster: variation in mating preference and correlation between sexes. Evolution 51: 11751181.
  • Houpt, D.R., Pursey, J.C. & Morton, R.A. 1988. Genes controlling malathion resistance in a laboratory-selected population of Drosophila melanogaster. Genome 30: 844853.
  • Howard, D.J. & Berlocher, S.H. (eds) 1998. Endless Forms: Species and Speciation. Oxford University Press, New York, USA.
  • Kilias, G. & Alahiotis, S.N. 1982. Genetic studies on sexual isolation and hybrid sterility in long-term cage populations of Drosophila melanogaster. Evolution 36: 121131.
  • Lande, R. 1981. Models of speciation by sexual selection on polygenic traits. Proc. Natl. Acad. Sci. USA 78: 37213725.
  • Lindsley, D.L. & Zimm, G.G. 1992. The Genome of Drosophila melanogaster. Academic Press, Inc, San Diego, CA, USA.
  • Maitra, S. 2000. Molecular and Genetic Analysis of Cytochrome P450 Gene Regulation in Drosophila melanogaster. PhD Dissertation. University of Tennessee, TN, USA.
  • Maitra, S., Dombrowski, S.M., Basu, M., Raustol, O., Waters, L.C. & Ganguly, R. 2000. Factors on the third chromosome affect the level of Cyp6a2 and Cyp6a8 expression in Drosophila melanogaster. Gene 248: 147156.
  • Maitra, S., Dombrowski, S.M., Waters, L.C. & Ganguly, R. 1996. Three second chromosome-linked clustered Cyp6 genes show differential constitutive and barbital-induced expression in DDT-resistant and susceptible strains of Drosophila melanogaster. Gene 180: 165171.
  • Markow, T.A. 1981. Mating preferences are not predictive of the direction of evolution in experimental populations of Drosophila. Science 213: 14051407.
  • Markow, T.A. 1991. Sexual isolation among populations of Drosophila mojavensis. Evolution 45: 15251529.
  • Merrell, D.J. & Underhill, J.C. 1956. Selection for DDT resistance in inbred, laboratory, and wild stocks of Drosophila melanogaster. J. Econ. Entomol. 49: 300306.
  • Orr, H.A. 2001. The genetics of species differences. Trends Ecol. Evol. 16: 343350.
  • Rice, W.R. & Hostert, E.E. 1993. Laboratory experiments on speciation: what have we learned in 40 years? Evolution 47: 16371653.
  • Ringo, J., Wood, D., Rockwell, R. & Dowse, H. 1985. An experiment testing two hypotheses of speciation. Am. Nat. 126: 642661.
  • Shaw, K.L. 1996. Polygenic inheritance of a behavioral phenotype: interspecific genetics of song in the Hawaiian cricket genus Laupala. Evolution 50: 256266.
  • Shufran, R.A., Wilde, G.E. & Sloderbeck, P.E. 1997. Life history study of insecticide resistant and susceptible greenbug (Homoptera: Aphididae) strains. J. Econ. Entomol. 90: 15771583.
  • Stevens, L. 1994. Genetic analysis of cannibalism behavior in Tribolium flour beetles. In: Quantitative Genetic Studies of Behavioral Evolution (C. R. B.Boake, ed.), pp. 206227. University of Chicago Press, Chicago, USA.
  • Sundseth, S.S., Kennel, S.J. & Waters, L.C. 1989. Monoclonal antibodies to resistance-related forms of cytochrome P450 in Drosophila melanogaster. Pestic. Biochem. Physiol. 33: 176188.
  • Ting, C.-T., Takahashi, A. & Wu, C.-I. 2001. Incipient speciation by sexual isolation in Drosophila: concurrent evolution at multiple loci. Proc. Natl. Acad. Sci., USA 98: 67096713.
  • Turner, G.F. & Burrows, M.T. 1995. A model of sympatric speciation by sexual selection. Proc. R. Soc. Lond. B 260: 287292.
  • Underhill, J.C. & Merrell, D.J. 1966. Fecundity, fertility, and longevity of DDT-resistant and susceptible populations of Drosophila melanogaster. Ecology 47: 140142.
  • Waters, L.C. & Nix, C.E. 1988. Regulation of insecticide resistance-related cytochrome P-450 expression in Drosophila melanogaster. Pestic. Biochem. Physiol. 30: 214227.
  • Waters, L.C., Zelhof, A.C., Shaw, B.J. & Ch'ang, L.-Y. 1992a. Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila. Proc. Natl. Acad. Sci. USA 89: 48554859.
  • Waters, L.C., Shaw, B.J. & Ch'ang, L.-Y. 1992b. Regulation of the gene for Drosophila P450-B1, a P450 isozyme associated with insecticide resistance. In: Molecular Mechanisms of Insecticide Resistance: Diversity Among Insects (C. A.Mullin & J. G.Scott, eds), Symposium series no. 505, American Chemical Society, pp. 4252. Oxford University Press, Oxford.
  • Wu, C.-I., Hollocher, H., Begun, D.J., Aquadro, C.F., Xu, Y. & Wu, M.-L. 1995. Sexual isolation in Drosophila melanogaster: a possible case of incipient speciation. Proc. Natl. Acad. Sci. USA 92: 25192523.

Received 24 July 2002;accepted 13 August 2002