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Factors Affecting the Distribution of Cytoplasmic Incompatibility in Drosophila simulans

Ary A. Hoffmann,” Michael Turellit and Lawrence G. Harshman*

*Department of Genetics and Human Variation, La Trobe University, Bundoora, 1083 Australia, tDepartment of Genetics and ‘Department of Entomology, University of California, Davis, California 95616

Manuscript received February 23, 1990 Accepted for publication August 10, 1990

ABSTRACT In Drosophila simulans a Wolbachia-like microorganism is responsible for reduced egg-hatch when

infected males mate with uninfected females. Both incompatibility types have previously been found in North America, Europe and Africa. Some California populations have remained polymorphic for over two years, and the infection is apparently spreading in central California. Egg hatch proportions for wild-caught females from polymorphic populations show that the incompatibility system acts in nature, but egg mortality rates are apparently lower than observed in laboratory populations. Although infected females maintained under various laboratory conditions never produce uninfected offspring, some wild-caught infected females produce both infected and uninfected progeny. This helps explain the persistence of a low frequency of uninfected flies in predominantly infected populations and may also explain the other polymorphisms observed. Fitness comparisons of infected and uninfected stocks, including both larval and adult fitness components, indicate that fecundity may be the component most affected. Infected females suffer a fecundity reduction of IO-20% in the laboratory, but the reduction seems to be smaller in nature. A theoretical analysis provides some insight into the population biology of the infection.

C YTOPLASMIC incompatibility is widespread in insects, and was first described in Drosophila

simulans by HOFFMANN, TURELLI and SIMMONS (1 986) in crosses between Californian populations. The lines previously described fell into two types, termed “R” and “W” (HOFFMANN and TURELLI 1988). W females are incompatible with R males, in the sense that a significant fraction of the eggs produced by such matings fail to hatch. In contrast, the reciprocal cross and crosses within compatibility types all produce comparable numbers of progeny. Laboratory studies indicate that type R mothers produce only type R offspring, whereas matings between R males and W females produce 1-2% R offspring (HOFFMANN and TURELLI 1988). Type R strains can be converted to type W by culturing them on medium containing tetracycline, suggesting that incompatibility is caused by a microbial infection in the R strains. In support of this hypothesis, LOUIS and NICRO (1 989) and BIN- NINGTON and HOFFMANN (1989) found that a Wolba- chia-like microorganism is present in the gonadal tis- sue of R strains but absent from W strains.

Both incompatibility types appear to be widespread throughout the world, and have been recorded from Africa and Europe as well as the east and west coasts of North America (HOFFMANN and TURELLI 1988; our unpublished data). In contrast, extensive sampling has not uncovered R strains in Australia. Both incompatibility types can also be found in collections from

<;cne[ic.s 126: 933-948 (December, 1990)

the same site, at least in California (HOFFMANN and TURELLI 1988) and France (J. R. DAVID, personal communication). Polymorphism is not predicted by simple models for the population dynamics of incompatibility factors. If cytoplasmic incompatibility is unidirectional, such that uninfected females are incompatible with infected males, uninfected females will produce fewer adult offspring than infected females, and their disadvantage increases as the infection be- comes more common. Thus, the infection is expected to spread from its center of origin and polymorphisms for the two types should not persist (CASPARI and WATSON 1959).

Our population data suggests that other factors must contribute to the frequency of incompatibility types. Some possibilities are summarized in Table 1 and are addressed by the experiments presented below. Perhaps the simplest explanation for persistent polymorphism is that incompatibility is expressed in the laboratory but not under field conditions. We have tested this possibility by scoring egg hatch for wild-caught R and W females from polymorphic populations.

A second possibility is that under field conditions infected females produce both infected and uninfected progeny. We have no evidence of such “segregation” in the laboratory, except in a single line associated with paternal transmission of the infection (HOFFMANN and TURELLI 1988), but several factors

934 A. A. Hoffmann, M. Turelli and L. G. Harshman

TABLE 1

Factors that may help to explain the patchy geographical distribution and polymorphism of incompatibility types in D.

simulans

1 .

2.

3.

4. 5.

6.

7 .

Incompatibility between R and W types is not expressed under

Occasional W progeny from R mothers a. Curing of infection by naturally occurring antibiotics b. Curing of infection by exposure to extreme temperatures Deleterious viability or fecundity effects of the incompatibility-

Nonrandom mating of incompatibility types Effects of incompatibility type on female remating speed or

Migration between populations nearly monomorphic for R or

Interactions between nuclear eenes and the infection

field conditions

causing infection

sperm competition

w

that we have not tested may cause segregation in the field. In particular, exposure to high temperatures may be involved because high temperatures suppress incompatibility (HOFFMANN, TURELLI and SIMMONS 1986); and age may be involved because old R males tend to have fewer Wolbachia-like organisms and are more compatible with W females (HOFFMANN, TUR- ELLI and SIMMONS 1986; BINNINCTON and HOFFMANN 1989). Segregation may also occur when larvae encounter naturally occurring antibiotics, as demon- strated in Tribolium (STEVENS 1989). We have tested for segregation in the laboratory and with field-collected females.

Third, the dynamics of the infection may be influenced by deleterious effects on fitness. Theoretical analyses show that such effects produce an unstable equilibrium frequency, below which the infection is lost, but above which it spreads to fixation (CASPARI and WATSON 1959; FINE 1978). We have previously found suggestive evidence for deleterious fitness effects by comparing the number of progeny produced by R strains to the number produced by derivative strains treated with tetracycline to cure the infection (HOFFMANN and TURELLI 1988). These results are only suggestive because tetracycline may affect fitness- related factors other than the incompatibility system. Also, we did not previously examine fitness components other than productivity. We have therefore carried out tests on a wider range of fitness components using comparisons other than those involving tetracycline-treated strains, and we have also obtained fecundity data from co-occurring R and W flies from the field.

A fourth possibility is that incompatibility dynamics are influenced by mating behavior or sperm competition. Mating between R and W flies may be nonrandom; for example, W females may avoid mating with infected males. We have presented evidence against this in virgin matings (HOFFMANN and TUR-

ELL1 1988); here we describe experiments with previously inseminated females, because such females mate less readily and may therefore be more discriminating. Incompatibility dynamics may also be influenced by other aspects of mating that affect fitness. W females may remate more quickly than R females after mating with R males. Previous experiments with Drosophila melanogaster have found that the tendency of females to remate depends on properties of both sexes (PYLE and GROMKO 1981). Remating speed is particularly dependent on the amount of sperm stored by D. melanogaster females (GROMKO, NEWPORT and KOR- TIER 1984). Thus, if the Wolbachia-like infection affects sperm production or transfer, it may influence remating in D. simulans. Incompatibility dynamics could also be altered by differential sperm competition if the incompatibility mechanism involves factors that act prior to syngamy. We have therefore considered the remating dynamics of R and W females and the effects of prior mating with an R or W male on the progeny produced after a second mating.

Two other factors not considered in our experiments may also influence the distribution of incompatibility types. These are migration and nuclear- cytoplasmic interactions. Migration may account for persistent polymorphisms of incompatibility types if there is an unstable equilibrium frequency within populations and populations nearly fixed for the alternative types exchange relatively few migrants (4. KARLIN and MCGRECOR 1972; NICRO and PROUT 1990). Nuclear factors may reduce the level of incompatibility in crosses between W females and R males, although we have so far failed to find evidence for nuclear-cytoplasmic interactions. Both of these factors are considered in the DIxUSSION.

Before describing experiments concerning these factors, we briefly consider some recent California population surveys that supplement the data on the distribution of R and W in HOFFMANN and TURELLI (1 988). We also present data on the frequency dynamics of the incompatibility types in laboratory populations set up with lines from a persistently polymorphic field population.

MATERIALS AND METHODS

Stocks and field collections: Several collections at different times were made from citrus groves near Piru in a valley separated by the Santa Susana mountains from the Los Angeles basin in southern California. Flies were collected by placing buckets with banana bait in the groves and netting the adults after a few hours or by collecting flies directly from fallen fruit. Collections were also made at a campground near Lake Cachuma, in the Santa Ynez mountains just north of Santa Barbara in southern California. Flies from these sites were used to examine field incompatibility levels and field segregation of incompatibility types. A few other sites in southern and central California were sampled to obtain additional data on the spatial and temporal fre-

935 Incompatibility in Drosophila

TABLE 2

Designation of stocks used in the laboratory experiments

Code Origin Description

R stocks RH Highgrove 1985 Massbred from 18 isofemale lines RP Piru 1988 Massbred from 20 type R isofemale lines RPM Piru/Melbourne RP backcrossed to WM males for 5 generations RR Riverside 1984 Massbred from 30 isofemale lines R R W Riverside 1984 White eye stock derived from a wild-caught white eyed female Rscgs) Sanger 1985 Isofemale line

W stocks WH(J3) Highgrove 1985 Isofemale line WHT Highgrove 1985 Derived from R H by tetracycline treatment WM Melbourne 1985 Massbred from 25 isofemale lines WP Piru 1988 Massbred from 20 type W isofemale lines W P M Piru/Melbourne W p backcrossed to WM males for 5 generations WRT Riverside 1984 Derived from RR by tetracycline treatment W R T ( w ) Riverside 1984 Derived from R R ~ ~ ) by tetracycline treatment WS Sanger 1985 Massbred from 18 isofemale lines ww Watsonville 1984 Massbred from 30 isofemale lines

quency distribution of the incompatibility types. Several different stocks were used in the laboratory ex-

periments, and these are listed in Table 2 together with their designations. We established two independent R stocks (RIB) and two independent W stocks (Wp) from Piru. Each was initiated with the progeny of 20 isofemale strains from the November 1988 collection. We also used several of the massbred stocks described in HOFFMANN and TURELLI (1988). These include the R stocks Riverside (RR) and High- grove (RH), and the W stocks Melbourne (WM) and Sanger (Ws). We also used an R isofemale strain from Sanger (RS(LS)) and a W isofemale strain from Highgrove (WH(SS)), referred to as S13 and Hg33, respectively, in HOFFMANN and TUR- ELLI (1988). Finally, we performed remating experiments with a white-eye mutant stock, RR+), isolated from a River- side field collection in 1984. Other stocks were derived from these by treating R stocks with tetracycline to cure the infection and by backcrossing to WH males. All flies were cultured on standard Drosophila medium at 20-23" unless otherwise stated.

Frequency of incompatibility types: We sampled the population at Piru on four occasions over 3 years, while most other sites were sampled only once or twice. Females from the field were set up individually in vials. The incompatibility type of the resulting isofemale line was determined by pair mating 3-5 male progeny to virgin type W females (W, or Ww) and 3-5 virgin female progeny to type R males (RR). Pairs of flies 0-3 days old were placed in vials and left to oviposit for 2-3 days. For vials with at least ten eggs, we scored the proportion of eggs that hatched 25-48 hr after the adults were removed. Crosses were considered incompatible when <25% of the eggs hatched, and compatible when >75% hatched. In hundreds of crosses, we have found that more than 95% of our control crosses with R and W stocks maintained in the laboratory produce unambiguous results with this criterion. Exceptions almost always involve compatible crosses that produce egg-hatch rates below 75%. Results from newly established isofemale lines are generally consistent for the two types of crosses, L e . , egg hatch is consistently low either when the strain females are mated to R males or when the strain males are mated to W females. We have found no strains in which either both or neither of these crosses produced consistently low egg hatch. How- ever, we have found some newly established isofemale lines

that yield inconsistent results, i .e., the replicate crosses produce inconsistent results or produce intermediate (25-75%) proportions of hatched eggs. These are not counted in our estimates of R and W frequencies but are discussed sepa- rately.

Incompatibility levels in the field The first experiment was carried out with females collected at Piru and Lake Cachuma in July 1988. Females and males were taken to the laboratory after having been stored in vials for 1-2 days following the field collection. Females were set up individually on spoons containing grape juice concentrate and Drosophila medium, and covered with a live yeast suspen- sion. Females were transferred to fresh spoons after 24 hr. After another 24 hr of egg laying, females were transferred to standard food vials to establish isofemale lines. The number of hatched and unhatched eggs on each spoon was scored >24 hr after the female was removed. Each isofemale line was characterized for incompatibility type as described above.

In the second and third experiments (November 1988 and July 1989), flies were only collected from Piru. The sexes were separated in the field immediately after collection to prevent mating in the storage vials. Remating in storage vials could have biased the estimate of incompatibility in the first experiment, because compatibility is increased as R males get older.

In each of these experiments, we protected the flies from heat stress during transport to the laboratory by shielding them from the sun and using air conditioning in the summer. During our summer collections, the temperature at the collection sites approached 37'. Our laboratory tests were performed at room temperature, 20-23 O .

Incompatibility changes in laboratory populations: We used the mass-bred Rp and Wp stocks from Piru for this experiment. Each population was initiated with 30 males and 30 females of each incompatibility type. Populations were maintained in 600-ml bottles with 50 ml of medium. Ten replicate populations were maintained by transferring 60 adults that were 0-1 day old to fresh medium each generation, and removing the adults after 3 days. A second set of ten populations was maintained by collecting adults that were 0-1 day old and holding them for 8 days before setting up bottles for the next generation. Incompatibility frequencies were tested after five and ten generations. Fifty


virgin females from each population were crossed to RK males, and incompatibility type was determined from the proportion of the eggs that hatched. We also included crosses of Rp males to Rp females to determine the percent- age of vials that may have appeared incompatible because of poor hatchability not associated with incompatibility.

Effects of male age: Previous experiments had indicated that the age of R males influenced levels of incompatibility, while the age of W females had no effect (HOFFMANN, TURELLI and SIMMONS 1986). We examined the effect of male age and temperature in more detail by crossing Rp males that were 1-24 days old to Wp females that were 2- 6 days old. In the first experiment, males were collected every day for 24 days and aged at 20" before crossing. In the second experiment, males were aged at 28" and only males aged for 1, 3, 6, 9, 12, 15, 18 and 21 days were used. T o determine incompatibility, pairs of males and females were placed in vials with a spoon as described above for the characterization of field incompatibility levels, except that the grape-juice medium was replaced by a black currant medium. Twenty replicate vials were set up for each male age class. Control crosses (W, X Wp and Rp X Rl,) were also set up in a separate experiment with males that were 1, 12 and 24 days old (20") or males that were 1, 12 and 21 days old (28"). Males were crossed to females that were 2-6 days old and ten replicates were set up for each cross.

Segregation experiments: Three different experiments were performed to determine conditions under which R females may produce W offspring.

High temperature: RK flies were cultured at 28", a temperature that causes a temporary suppression of incompatibility (HOFFMANN, TURELLI and SIMMONS 1986). Female progeny were collected as virgins and crossed to WM males. These flies were placed at 28" again, and F1 progeny were backcrossed to WM males. This procedure was repeated for another three backcross generations. Any type W female progeny that emerge should persist in the population because they are compatible with WM males. T o test for segregation at the end of the experiment, females were set up individually in vials and progeny were tested for incompatibility type at room temperature as described above.

Aged type R females: At room temperature, compatibility is restored to a large extent when R males older than 2 weeks are crossed to type W females (HOFFMANN, TURELLI and SIMMONS 1986), suggesting that the level of infection may be reduced. This could also occur in old females and may lead to segregation. Using R R flies, we therefore set up 198 isofemale lines from the progeny of 30 females 3 weeks old, as well as 196 isofemale lines from females 2-4 days old. Progeny were crossed to WM males in establishing the isofemale lines to ensure that any type W segregants would produce offspring.

Field segregation: In the third Piru collection, progeny from field-collected females that had been identified as type R were tested for incompatibility type by crossing females to RK males. We tested 10 progeny from 44 females and established an isofemale line for each of the progeny to verify their incompatibility type. We also tested lines derived from a female (designated 34) that could not be assigned to an incompatibility type because the level of incompatibility in crosses with R K males was intermediate.

Fitness experiments: T o examine the effects of incompatibility type on fitness and mating, we attempted to compare R and W stocks with similar nuclear backgrounds. In earlier experiments (HOFFMANN and TURELLI 1988), we compared stocks that had been cultured for a generation on medium containing tetracycline (0.03%) with parental R stocks that were not exposed to tetracycline. The antibiotic

treatment converts R stocks to W stocks (HOFFMANN, TUR- ELLI and SIMMONS 1986), but may alter cellular components other than the incompatibility factor. In an alternative approach, R and W stocks were crossed reciprocally, and fitness comparisons were made between the Fls. The incompatibility factor is largely inherited via the maternal parent (HOFFMANN and TURELLI 1988), so that Fls will have the incompatibility type of their maternal parent. The nuclear background of the FI females will be identical, although males will have an X chromosome and the mitochondrial from the maternal stock. Our third approach involved comparisons of the Piru stocks Rp and Wp. Replicate R and W stocks from Piru had been set up from different sets of isofemale lines. These stocks should have similar nuclear backgrounds; but they differ in their mitochondrial ge- nomes because of an association between mtDNA variation and incompatibility type in this population (HALE and HOFF- MANN 1990). In a fourth approach, we made the nuclear background of R and W lines similar by backcrossing lines to the same laboratory stock for several generations.

Egg-to-adultj tness components: These experiments tested the effects of incompatibility type on egg-to-adult viability, development time and biomass of the emerging adults. Comparisons were made between tetracycline-treated and untreated lines from the Riverside (RR) and Highgrove (RI,) stocks, and between the mass-bred RP and Wp lines from Piru. The tetracycline-treated stocks were obtained by culturing flies for one generation on medium with tetracycline as described in HOFFMANN and TURELLI (1988), followed by two or three generations on medium without tetracycline. The treated stocks will be referred to as WKI. (Riverside) and WkI . , (Highgrove). Two WK-r lines and two WH,I. lines were set up after tetracycline treatment, and the fitness of these lines was compared to two RK or two REI lines set up at the same time. Twenty eggs from a line were transferred to vials containing 10 ml of laboratory medium. Twenty vials were set up per line. Adult progeny were collected daily and held in vials until all progeny had emerged, and flies were then weighed on a microbalance.

Fecundity: The effect of incompatibility type on fecundity was examined by comparing tetracycline-treated and untreated lines from Riverside used in the egg-to-adult fitness experiment. We also tested R and W stocks from Piru that had been backcrossed to Melbourne males for five generations, these are referred to as RpM and WPM, respectively. Males and females were collected as virgins within a 12-hr period. These flies had been cultured at a low density by placing groups of 20 eggs in vials with 10 ml of medium. R and W males and females were crossed in all possible combinations so that any fecundity effects could be assigned to male and female incompatibility types. Because two replicate R and W stocks were tested in each comparison, there were 16 combinations. Twenty replicate pairs of males and females were crossed for most combinations, although some vials were discarded because of mortality or escape during transfer.

The following procedure was used to reduce the variance in egg counts between vials. Pairs of males and females were left for two days in vials prepared without live yeast. Vials were placed on a flat surface with a white background under fluorescent light. Flies were then transferred to vials with 10 ml of a dark molasses medium that facilitates egg counting. The medium was covered with a layer of live yeast to induce oviposition. Vials were placed on a flat surface under dim light (approximately 30 lux). Flies were transferred to fresh vials after 24 hr and cleared after a second 24-hr laying period. Egg counts were pooled over the 2 days. The experiment was run in a randomized block design with

Incompatibility in Drosophila 937

FIGURE 1 .-Map of California indicating collection sites for samples in Table 3.

combinations randomized within the 20 blocks. Mating experiments. Two different experimental re-

gimes were used to assay different aspects of mating per- formance.

Muting success in large cages: Experiments were carried out in Perspex cubic cages 400 mm on a side. Two sides of the cages were open and covered with gauze. Eight plastic cups with laboratory medium were placed at the bottom of each cage. Rand W lines were tested pairwise. Two hundred males and 200 females were released into each cage. These flies had been aged together for 3 days at a density of 20- 30 flies per vial. The two lines to be tested were marked with a different color (green or pink) of micronized fluorescent dust prior to release. Flies were introduced into cages in the evening and left to settle for 12 hr. Mating pairs were removed at 1-hr intervals for a 12-hr period and were identified under UV light by their dust colors. We tested four pairs of R and W stocks. We compared the Fls from reciprocal crosses between Rand W lines from Sanger (Rs(ls) and Ws) and Highgrove (RH and WH(33)) as well as Fls from crosses between RR and WM. We also compared the mating success of Rp and Wp.

Remating success: To examine the effect of male incompatibility type on remating behavior, approximately 200 W females were first mated to R or W males when flies were 2-3 days old. Females were individually mated in vials so that males could be removed within 30 min after copulation was completed. Females that mated were held in vials with laboratory medium and live yeast for 2 days. This medium stimulates oviposition which increases the receptivity of females for remating. Females were paired with a second R or W male in fresh vials and observed for 12-14 hr, at which stage about half had remated. The effect of male incompatibility type was examined by comparing the number of females that had or had not remated for each combination of first and second males. This experiment was carried out with the R and W Fls from reciprocal crosses between R and W stocks established from single populations: the Sanger stocks (W, and RXls)), and the Highgrove stocks (RH and WH(s3)). It was also carried out with the mass-bred

TABLE 3

Distribution of R and W lines in 1987-1989 collections from California

Date Location #R #W freq(R) 95% CI“

6/1987 Highgrove 26 1 0.96 (0.81, 0.999) Ventura 10 2 0.83 (0.52, 0.98) Lake Cachuma 7 12 0.37 (0.16, 0.62) Piru 9 8 0.53 (0.28, 0.77) Arvin 0 20 0.00 0.14 Sanger 0 7 0.00 0.35

7/1988 Lake Cachuma 47 57 0.45 (0.35.0.55) Piru 38 14 0.73 (0.59, 0.84)

11/1988 Highgrove 9 0 1.00 0.72 Piru 76 97 0.44 (0.36, 0.52) Davis 0 12 0.00 0.22

7/1989 San Diego 38 2 0.95 (0.83, 0.99) Highgrove 245 15 0.94 (0.91, 0.97) Piru 44 31 0.59 (0.47, 0.70)

9/1989 Sanger 28 77 0.27 (0.19, 0.36) 11/1989 Ventura 13 1 0.93 (0.66, 0.998)

Arvin 19 46 0.29 (0.19, 0.42)

‘ “Exact” confidence intervals with probability 0.025 in each ta i l were computed directly from the binomial. When freq(R) equals 0.0 (1.0). the upper (lower) 5% value is given.

Rp and Wp stocks from Piru. Thus flies from predominantly W, predominantly R, and highly polymorphic populations were analyzed. The W Fls were obtained by crossing old R males to W females ($. HOFFMANN and TURELLI 1988).

Sperm competition: We used a Riverside stock homozy- gous for a recessive white eye gene (RR(J to investigate the effects of incompatibility type on sperm competition. White eye flies that were type W (WRT,,)) were obtained by tetracycline treatment. White eye virgin females that were type W were mated to wild type males (2-3 days old) that were either type W (WRT) or R (RR). The WRT males were from the Riverside stock that had been treated with tetracycline for one generation. Males and females were set up in pairs so that mating could be monitored. Males were removed within 30 min after we observed mating. Females were then held individually in vials with live yeast for 4 days before they were exposed to a white eye male that was type R (RR(,,,)) or W (WRT(,)). Most of the females (>60%) were observed to remate within 12 hr. Males were removed within 30 min after we observed remating and females were transferred to fresh yeasted vials. Females were subse- quently transferred to new vials every 3 days until no more progeny were produced. The number of white eye and wild type progeny that emerged from eggs laid after the second mating were scored.

RESULTS

Frequency of incompatibility types in 1987-1989 collections: The number of R and W lines from various California locations as well as confidence intervals for the frequency of R are given in Table 3. The approximate locations of these samples are indicated in Figure 1. The 1987 collection indicates that the Lake Cachuma and Piru locations are polymorphic and both incompatibility types are present at intermediate frequencies. These polymorphisms are not transient because they persist in the 1989 Piru collec-

938 A. A. Hoffmann, M . Turelli and L. G. Harshman

tions. However, a contingency test indicates significant heterogeneity in the four Piru collections (G = 15.48, d.f. = 3, P < 0.002); and the difference between the two 1988 collections was also significant (G = 14.02, d.f. = 1) even though they were separated by only 4 months.

We failed to collect any R strains in the Central Valley in 1987, but we collected several R strains in large samples from Sanger and Arvin in 1989. The confidence intervals indicate that the frequency distribution of R and W was not significantly different in the 1987 and 1989 Sanger collections, but the 1987 sample is quite small. However, the 1989 Sanger collection does differ significantly from the combined 1987 Central Valley collections from Arvin and San- ger (G = 14.64, d.f. = 1, P < 0.001). The 1987 and 1989 Arvin collections do differ significantly (G = 11.78, d.f. = 1 , P < 0.001), but the 1989 collections from Arvin and Sanger do not.

In agreement with earlier results (HOFFMANN and TURELLI 1988), we found a low frequency of W strains in the predominantly R populations at Highgrove and Ventura. A 1989 sample from San Diego gave similar results. There are no statistically significant differences among the six samples from Highgrove, Ven- tura and San Diego. Pooling them, we find that the frequency of R in these southern California populations is 0.94, with a 95% confidence interval of 0.91- 0.96.

In the 1989 Arvin sample, 83 isofemale lines were successfully tested using the F1 from field-collected females. For each line, Si, we set up three replicates of each test cross, Si? X RRd and WW? X Sid. From these, 65 lines could be assigned to an incompatibility type using the criteria that all replicates of each cross were consistent, and only one of the two types of crosses was compatible. The remaining 18 lines could not be assigned to an incompatibility class because of inconsistency among the replicates. Isofemale sublines have been set up from these ambiguous lines for further analysis.

Incompatibility in the field: In the first and fourth field collections, most females laid only a few eggs in the first 24-hr period and counts were pooled over the 2 days. In the second and third field collections, most fenlales laid more than ten eggs in the first 24- hr period, and egg counts were therefore not made in the second laying period. One-tailed t-tests were used to test for a deleterious effect of the infection on fecundity. The proportion of eggs that hatched showed a skewed distribution because all eggs hatched in many cases. Non-parametric Kruskal-Wallis tests were therefore used to test for a decrease in the proportion of hatched eggs in vials with W females. Proportions were calculated only for females that laid ten or more eggs.

There was no statistically significant effect of incompatibility type on the proportion of eggs that hatched in the Lake Cachuma collection (Table 4). In contrast, W females produced significantly higher proportions of unhatched eggs in all three Piru collections, with differences in the proportion unhatched ranging from 13% to 28%. These results provide clear evidence that cytoplasmic incompatibility is expressed under field conditions at Piru.

We can use the hatch rates for field-collected females to estimate the relative hatch rate produced by incompatible ( i e . , W? X Rd) vs. compatible crosses (ie., R X R, W X W, or R? X Wd). Assuming that all three compatible crosses produce the same average hatch rate, let Hr; denote the average hatch rate from compatible crosses, and let HI denote the average hatch rate from incompatible crosses. If the two incompatibility types mate at random in the field, the relative hatch rate from incompatible crosses can be estimated as

where flu. and RK denote the average hatch rates from field-collected W and R females and p denotes the frequency of R in the population. This expression holds irrespective of multiple mating, it simply requires that the fraction of eggs fertilized by R sperm is p , the fraction of R adults. Table 5 presents two estimates of H for each field experiment as well as approximate 95% confidence intervals based on the bias-corrected percentile method with 1000 bootstrap samples (EFFRON 1982, Sec. 10.7). The two estimates of H for each experiment result from computing the averages RW and fl, in (1) in different ways. The “unweighted averages” are obtained by calculating a hatch rate for each female that produced ten or more eggs, then averaging these hatch rates for R and W females. The “weighted averages” are obtained by pooling all the eggs laid by females of the same incompatibility type (irrespective of the number laid), then calculating the fraction of all of these that hatched. As shown in Table 5, both averages give very similar results.

Turning to the number of eggs laid by females, the mean number for W females was greater than the mean number for R females in all four collections, but the difference was only significant for the Lake Cachuma collection. Our field data therefore provide no evidence for consistent deleterious effects of the infection on fecundity. Approximate power calcula- tions indicate that we could have detected a difference of 2.30 eggs, or a deleterious effect of 8%, with probability 0.5 in the largest collection at Piru and a deleterious effect of 12% with probability 0.8. This suggests that deleterious effects are small if present.


TABLE 4

Incompatibility levels and number of eggs laid (with sample size N) by R and W females collected from the field

Type R T y p e w Collection rf(S0) N X(SD) N P

Lake Cachuma 7/88 Number of eggs 27.1 (13.8) 47 32.8 (17.3) 57 C0.05 Proportion hatched 0.95 (0.07) 42 0.91 (0.12) 52 NS

Number of eggs 40.6 (1 1.9) 38 41.2 (20.8) 14 NS Proportion hatched 0.95 (0.06) 38 0.82 (0.20) 14 co.001

Number of eggs 29.0 (9.8) 76 29.9 (8.5) 97 NS Proportion hatched 0.98 (0.05) 75 0.77 (0.29) 96 co.001

Number of eggs 42.0 (16.8) 44 46.6 (14.4) 31 NS Proportion hatched 0.97 (0.04) 41 0.69 (0.36) 31 co.001

Piru 7/88

Piru 1 1/88

Piru 8/89

Probabilities are for one-tailed t-tests (number of eggs) or Kruskal-Wallis tests (proportion hatched).

TABLE 5

Inferred relative egg hatch proportions (H) from incompatible matings in the field

Unwei hted H Weighted H Collection ( 9 5 8 CY) (95% CI)

Lake Cachuma 7/88 0.91 (0.83, 1.00) 0.91 (0.82, 0.99) Piru 7/88 0.81 (0.63, 0.95) 0.85 (0.72, 0.97) Piru 11/88 0.52 (0.33, 0.66) 0.53 (0.34, 0.68) Piru 8/89 0.50 (0.22, 0.71) 0.57 (0.29, 0.77)

Based on the bias-corrected percentile method with 1000 bootstrap samples. If we weight each of these estimates by the inferred number of incompatible m_atings, the average H using weighted (unweighted) estimates of HW and HR equals 0.65 (0.67) with the Lake Cachuma data and 0.56 (0.59) without the Lake Cachuma data.

The relative productivities (numbers of eggs laid) for R vs. W females in the four samples range from 0.83 to 0.985 and average 0.92.

Changes of incompatibility-type frequency in laboratory populations: These experiments tested whether type R increased in frequency as expected when populations were started at intermediate frequencies similar to those found at Piru, using the mass-bred Rp and WP stocks from this location. When flies were not aged prior to being transferred to fresh bottles, the R incompatibility type increased in all ten populations after five generations, as shown by the increased frequency of compatible matings (Figure 2). In nine of the ten populations, an additional increase was observed after ten generations. For populations maintained with aged flies, eight of the ten populations showed an increase in the frequency of compatible matings after five generations. The two populations that did not show an increase were maintained for a further five generations, and the number of compatible matings increased to 92% and 86% after ten generations. We did not expect all crosses to be “compatible” even when R had gone to fixation, because vials with females that had not mated or that

”Ol

0.91

0.44 0 5 10

generation FIGURE 2.-Changes in the frequency of compatible crosses in

laboratory populations started with 50% R and 50% W flies from mass-bred Piru stocks, R p and Wp. The solid (open) squares indicate populations in which 0-1-day-old (8-9-day-old) flies were used to set up each generation. The initial frequency of compatible matings is estimated from the controls.

produced many inviable eggs would also be scored as incompatible. The control matings between Rp males and females indicated that 5-8% of vials from compatible matings had low hatchabilities, and this suggests that some of the populations were close to fixation for R after five generations in both sets of experiments. Based on these controls, we assumed that 6.5% of RP X Rp matings would be scored as incompatible when setting the initial frequency of compatible matings to 47% in Figure 2. The rapid increase of R in the aged-fly populations is perhaps surprising given the increase in compatibility with male age (see below). However, the flies were not aged as virgins; so this outcome may simply reflect a low frequency of remating after the females had mated with young males.


-.- 0 5 IO 15 20 25

0 5 10 15 20 25 male age (days)

FIGURE 3.-Fraction of eggs that hatch from Wp? X R& crosses set up with males of different ages. The solid squares indicate the means for these incompatible crosses. The open circles (squares) indicate the means for the Rp X Rp (Wp X WP) controls. The error bars are experimental standard deviations, not standard errors of the means.

Effects of male age and temperature: The proportion of eggs that hatched was calculated for all females that laid ten or more eggs. Proportions are plotted against male age in Figure 3. Data points are based on at least 12 females. The proportion of eggs that hatched increased with male age as expected. For males stored at 20 " , hatchability increased gradually and appeared to level out at 50-70% when males were two or more weeks old, although the error bars (which provide standard deviations, not standard errors for the means) indicate that the level of incompatibility varied enormously from vial to vial. For the males stored at 28", the proportion of eggs that hatched increased more quickly but appeared to level out at an earlier age at around 40%.

In the Wp X Wp control crosses with males stored at 20", males that were 1, 12 and 24 days old produced mean hatchabilities of 0.89 (SD = 0.1 l), 0.83 (0.17) and 0.81 (0.30), respectively, while the mean hatchabilities for the Rp X Rp control crosses with males that were 1, 12 and 21 days old were 0.9 1 (SD = 0.10), 0.86 (0.13) and 0.76 (0.22), respectively. Both controls suggest a decrease in hatchability when older males are used, although only the 1 us. 21-day- old comparison at 28" was statistically significant (P < 0.05). Figure 3 shows that the control hatch rates obtained with males that are 1 or 12 days old fall outside the experimental standard deviations obtained from the incompatible crosses, but those with the 24- day-old males do not. The Wp X Wp crosses with

males stored at 28" produced mean hatchabilities of 0.87 (SD = ().lo), 0.84 (0.12) and 0.64 (0.22), respectively for males that were 1, 12 and 2 1 days old; the corresponding figures for the Rp X Rp crosses were 0.87 (SD = 0.07), 0.80 (0.10) and 0.65 (0.26), respectively. The means for 21-day-old males fall within the empirical standard deviations of the means from the incompatible crosses, while means for the younger males do not (Figure 3).

These results suggest that egg hatchability in incompatible crosses approaches control levels when females are crossed to older males, although a low level of incompatibility probably persists even when males are 3 weeks old. Averaging the results for the Rp X Rp and Wp X Wp control crosses, the relative hatch rate for the incompatible crosses is 0.77 for 24-day-old males at 20" and 0.74 for 2 1-day-old males at 28". In contrast, with 1-day-old males, the relative hatch rate from incompatible crosses is 0.014 at 20" and 0.002 at 28", so that incompatibility is virtually complete; and with 12-day-old males, the relative hatch rate is 0.42 at both 20" and 28". Thus, once hatch rates are normalized by the controls, adult temperature seems to have little effect on incompatibility.

Segregation experiments: None of the laboratory experiments provided evidence for segregation of type W flies from the RR stock. In the high temperature experiment, all 200 lines established from flies after five generations at 28" were type R. In the experiment with old females, all of the 240 lines were type R, indicating that old RR females did not produce type W progeny. We have carried out other experiments using heat shock (35") and culture under cold temperatures with similar results. The incompatibility factor is therefore transmitted with complete fidelity via the maternal lineage under a range of conditions.

From our 1989 Piru collection, we identified four females out of 44, initially characterized as type R, that produced both R and W type progeny. These females produced a single W progeny from the 9-10 progeny that we tested. Isofemale lines were established from all progeny characterized as W, and the incompatibility type of these lines was determined from crosses to RR males and WM females in the next generation. All four lines were found to be W, as expected. However, when retested after another two generations, one of these lines was characterized as R. Further switches in incompatibility type were not detected in subsequent generations.

Progeny from female 34 could not be assigned an incompatibility type because some showed intermediate levels of compatibility when crossed to RR males. The offspring from these progeny were used to establish isofemale lines, which were characterized for incompatibility type in subsequent generations. When tested one generation after they were set up, five lines

Incompatibility in Drosophila 94 1

TABLE 6

Egg-to-adult fitness components for incompatibility types

Number of Development Weight per Comparison progeny time (days) fly (g x IO")

Highgrove (tetracycline) Means (SD)

RH(line 1 ) 16.3 (4.0) 12.74 (0.30) 10.96 (0.67) RH(line 2) 14.6 (4.0) 12.65 (0.28) 11.07 (0.68) WKT(line 1 ) 16.2 (4.5) 12.65 (0.34) 10.95 (0.74) WRT(line 2) 15.3 (4.5) 12.65 (0.35) 10.89 (0.69)

ANOVAs (mean squares) Incompatibility type (d.f. = 1) 2.00 0.24 0.17 Replicate line (d.f. = 2) 17.49 0.13 0.07 Error (d.f. = 70) 17.96 0.10 0.48

Riverside (tetracycline) Means (SD)

RR(line 1) 14.7 (4.2) 12.79 (0.36) 10.63 (1.31) RR(line 2) 17.6 (2.4) 12.54 (0.23) 10.91 (1.29) WRT(line 1) 16.6 (2.7) 12.82 (0.40) 11.07 (1.74) WRT(line 2) 16.8 (3.3) 12.47 (0.25) 10.51 (1.26)

ANOVAs (mean squares) Incompatibility type (d.f. = 1 ) 5.30 0.01 0.00 Replicate line (d.f. = 2) 42.41 0.92*** 1.94 Error (d.f. = 70) 10.50 0.10 1.99

Piru (mass bred) Means (SD)

Rp(line 1) 15.2 (1.7) Rp(1ine 2) 16.4 (2.1) Wp(line 1) 15.7 (1.8) Wp(line 2) 16.6 (2.1)

ANOVAs (mean squares) Incompatibility type (d.f. = 1) 3.10 Replicate line (d.f. = 2) 11.13 Error (d.f. = 75) 3.89

10.93 (0.72) 11.02 (0.68) 1 1.67 (0.76) 1 1.29 (0.65)

5.10 0.75 0.47

*** P < 0,001. Means are based on 20 vials.

were assigned as W and three lines were assigned as R on the basis of crosses to RR males and WM females. One of these W lines had switched to R when lines were retested two generations later. No further switches in incompatibility type were detected in later generations.

T o test for segregation in these flies under laboratory conditions, we pooled the R progeny from each of the four females that produced one W progeny to establish four lines. Thirty females were collected from each line and mated individually to WM males. Progeny from the females were tested for incompatibility type, and all were found to be R. This indicates that R females originating from Piru do not produce W progeny after several generations of laboratory culture, in agreement with earlier results with a Riv- erside stock (HOFFMANN and TURELLI 1988). This was confirmed by testing 100 isofemale lines from one of the mass-bred Rp stocks, all were found to be R.

Fitness experiments: Egg-to-adult-jtness: Results from these experiments are summarized in Table 6. ANOVAs indicate that there was no statistically significant effect of incompatibility type on any of these

fitness components, regardless of whether W lines from a tetracycline treatment or Wp lines were used in the comparison. Using a t-test, we find that these experiments had the power to detect a viability difference of about 10% (15%) with probability 0.5 (0.8) once data from the replicate lines are pooled. It should be noted, however, that our experimental regime, with 20 eggs per vial, provides minimal competition.

Fecundity: Mean egg counts for each combination of R and W lines (Table 7) suggest that R females produced fewer eggs than W females in comparisons with tetracycline-treated lines from from Riverside and with Piru lines backcrossed to WM males, regardless of the incompatibility type of the male. This was tested with mixed-model ANOVAs (Table 8) using the model

y . . y k l - - a + bi + cj + dh( i ) + et(j) + f j + gm + h z ( i j h l m ) ,

where a is the grand mean, bi the term due to female incompatibility type, c, the male incompatibility term, drc,) and el(,) the nested replicate line terms, f j the interaction between male and female incompatibility types, g , the block term and Itn(+,) the error term. Male and female incompatibility type were treated as

942 A. A. Hoffmann, M. Turelli and L. G . Harshman

TABLE 7 Mean fecundities (and standard deviations) from reciprocal crosses between R and W lines

Incompatibility type of males Incompatibility type of females

Tetracycline-treated stocks

RK(line 1) RK(line 2) WK.,(line 1) WKr(line 2)

RK(line 1) RR(line 2) WRT(line 1) WRT(line 2) 67.8 (13.3) 71.9 (21.5) 72.5 (24.4) 76.1 (13.5) 63.6 (14.3) 7 1 .O (1 3.6) 82.5 (1 1.7) 75.5 (16.8) 70.7 (18.8) 73.4 (1 3.9) 75.9 (18.2) 81.4 (15.7) 72.1 (17.9) 7 1.8 (20.3) 72.5 (15.5) 76.1 (11.7)

Piru lines (backcrossed) Rp(line 1) Rp(line 2) Wp(line 1) Wp(line 2)

R,.(line 1) 55.6 (20.3) 50.4 (20.3) 71.6 (10.6) 69.1 (14.9) R,.(line 2) 61.7 (20.3) 51.6 (21.3) 63.3 (20.1) 71.4 (13.0) Wp(line 1) 49.5 (24.5) 60.2 (19.8) 68.5 (17.6) 67.4 (15.4) Wp(line 2) 60.6 (13.7) 56.3 (27.8) 60.2 (23.6) 74.0 (19.0) Means are based on 17-20 vials.

TABLE 8

ANOVAs for fecundity data from reciprocal crosses between R and W lines

Mean squares

Effect Tetracycline-treated

d.f. lines Piru lines

Female type 1 2,917.1" 12,041.7' Male type 1 152.2 5.7 Repl. lines (female type) 2 251.7 450.2 Repl. lines (male type) 2 126.7 42.7 Block 19 410.3* 559.4 Female X male type 1 237.3 213.3 Error 2791282 246.5 367.0

* P < 0.05. a Female incompatibility type is not significant (P < 0.10) when

tested over replicate line term, but highly significant ( P < 0.001) when tested over pooled error and replicate line terms.

* Female incompatibility type is significant ( P < 0.05) when tested over replicate line term and highly significant ( P < 0.001) when tested over pooled error and replicate line terms.

fixed effects, while replicate line was treated as a random effect. The effect of female incompatibility type was significant when tested over the replicate line term in the Piru line comparison, and was mar- ginally significant in the tetracycline line comparison. T o increase the degrees of freedom in the denomi- nator, the replicate line term was pooled with the error term on the basis of the P > 0.25 criterion (SOKAL and ROHLF 198 1). The female incompatibility term was highly significant in both experiments when tested over the pooled error. There were no other significant effects in the ANOVAs apart from a block effect in the tetracycline line comparison. R females therefore had lower fecundity than W females regardless of which males they mated with. Fecundity of the Rp females was 18% lower on average than that of Wp females, while the fecundity of the RR females was 8% lower on average than WRT females.

Mating experiments: Mating success in large cages:

The number of matings obtained by R and W males in the large cages are given in Table 9 and were compared with x2 tests. Mating success did not differ between the incompatibility types except in one of the comparisons between the mass-bred Piru stocks, when Wp males were more successful than Rp males. How- ever, this trend was not apparent in the other tests with these stocks or in the F1 comparisons of the other stocks.

Deviations from random mating were examined by calculating G statistics for an association between male and female incompatibility type after organizing the counts into 2 X 2 tables. The G value was significant (P < 0.05) for one of the comparisons (Highgrove Fls), where there was a tendency for negative assortative mating. However, there was no evidence for assortative mating in the other six trials ( P > 0.10 for the G values), indicating that mating was generally random with respect to incompatibility type.

Remating success: Data for the experiments examin- ing the effect of the first and second male on the tendency of females to remate are given in Table 10. Counts were analyzed by log-linear models, initially fitting a model with the main effects of first male, second male and remating categories. This model is given by

ln($;k) = U + ai + b; + C k ,

where u is the mean of the logarithms of the expected frequencies, ai is the effect of the first male type, b; is the effect of the second male type and ck represents the remating category. The change in the log-likeli- hood ratio (AG), when the interaction between the first male type and remating is fitted to this model ( m i k ) , indicates the effect of the first male type on the remating tendency, and the same procedure can be used to indicate the significance of the second male type (bc;,). For the experiment with the Sanger stocks, AG was 2.42 and 0.56, respectively (d.f. = 1) when


TABLE 9

Mating success of males in large population cages indicated by number of males mating

R and W lines compared n w P

Sanger (Fls from Rs(1,) X WS) 41 50 NS

Highgrove (Fls from RH X WH(SS)) 75 56 NS Riverside/Melbourne (Fls from RR X W,) 43 41 NS

Piru (Rp us. WP) Rep. 1 32 74 CO.001 Rep. 2 3 3 4 2 NS

Rep. 3 47 41 NS

Rep. 4 47 38 NS

the first and second male interactions were fitted, and these changes were not significant at the 5% level. For the Highgrove stocks, AG was not significant for the first male interaction (AG = 1.04) but was significant ( P < 0.05) for the second male (AG = 3.91). Neither the first male (AG = 0.30) nor second male (AG = 0.19) interactions were significant in the experiment with the Piru lines.

These results suggest that the incompatibility type of the first male had no consistent effects on remating behavior. Females that engage in an incompatible mating are therefore not more likely to remate than females first mated to a compatible male. The only significant result was for a second male effect in the experiment with the Highgrove lines, when W males were more successful than R males. However this finding was not repeated in the other two experiments.

Sperm competition: Results are presented in Table 1 1. Statistical analyses involved comparisons of treat- ments by nonparametric Kruskal-Wallis tests rather than parametric tests because the presence of zero counts meant that distributions were skewed. As expected, WRT(w) females that were first mated to wild males that were type W (WR,) produced more wild- type offspring than females first mated to RR males (1 + 2 us. 3 + 4) in all three experiments. Similarly, WRT(w) females remated with white eye males that were type W (WRT,,)) produced more white eye progeny than females remated with RR(w) males (1 + 3 us. 2 + 4). These comparisons are both significant ( P < 0.001). Incompatibility was therefore present in remating females as well as in females mating for the first time.

TABLE 10

Remating speed of incompatibility types indicated by the number of females remated

First Male Second Male Remated Not remated

Sanger (FIS from Rsc1s) X WS) W W 15 25 W R 15 25 R W 18 23 R R 25 21

W W 29 15 W R 14 28 R W 18 26 R R 17 22

W W 23 17 W R 29 14 R W 2 2 10 R R 20 2 0

Highgrove (Fls from RH X WH(.W))

Piru (Rp versus Wp)

Interactions between R and W sperm are indicated by comparisons 1 us. 2 and 3 us. 4 for the wild-type progeny and the results for these comparisons were consistent in the three experiments. Neither comparison is significant ( P > 0.10), which means that the number of wild-type progeny did not depend on whether the second mating involved a WRT(w) male or an R R ( ~ ) male. Sperm interactions are also indicated by comparisons 1 us. 3 and 2 us. 4 for the white progeny. These comparisons are non-significant ( P > 0. lo), indicating that progeny numbers from the second male’s sperm did not depend on whether the female had mated initially to an RR male or a WRT male.

DISCUSSION

We will first summarize our experimental findings and compare them to previous results, then use some simple models to try to interpret the spatial and temporal frequency patterns of incompatibility types found in our California population samples.

Fitness experiments: Our laboratory tests provided no consistent evidence for egg-to-adult fitness component differences, nonrandom mating, differential male mating success or differential sperm competition between the two incompatibility types. In general, the

TABLE 11

Number of wild-type (+) and white eye (w) progeny from double matings between different incompatibility types

First Second + Progeny w Progeny

mating mating N Mean SD Mean SD

1 . WK.,. RR(W) 19 8.37 6.97 16.89 10.17 2. WR.,- WRT(x) 18 10.28 6.79 74.89 25.61 3. RR WmT(x) 15 2.40 3.76 13.53 11.04 4. RR R R W 22 2 .77 3.88 67.41 26.60


tests had sufficient power to detect differences on the order of 10% with probability 0.5. We did, however, find statistically significant reductions of 8% and 18% in the relative fecundities of R females in comparison to W females. The smaller value was obtained when comparing tetracycline-treated and untreated lines. NICRO and PROUT (1 990) compared the net “productivity” (including both viability and fecundity effects) of R and W strains from Italy and found 6-1 1 % lower productivity for their R strain. Similar comparisons between treated and untreated lines in Table 10 of HOFFMANN and TURELLI (1988) implied significant fitness reductions of 10-3 1 %, with an average reduction of 22%. These results suggest that R females may generally have significantly lower fecundity than W females, at least under laboratory conditions, and that fecundity may be the fitness component most affected by the incompatibility system. This is fortunate because only fecundity differences can be readily estimated with flies from nature.

Our data from field-collected flies suggest that R females suffer a smaller fecundity disadvantage in nature than seen in the laboratory. Although the R females were less fecund in all four experiments, only the 17% difference observed in the Lake Cachuma sample is statistically significant. However, in the largest of our three Piru collections, a fecundity difference of 12% would have been been detected with probability 0.8. Our data are consistent with the hypothesis that R females generally do suffer a fecundity disadvantage, but they suggest that the disadvantage i n nature is likely to be closer to 10% than 20%. We cannot be certain that the deleterious effects are produced by the incompatibility-causing infection. In the comparison of the Piru lines, incompatibility types may be associated with different mtDNA variants or other cytoplasmic Factors that affect fecundity. Tet- racycline treatment will probably not influence the mitochondrial genome, but it may influence other cytoplasmic factors that influence fitness. We found that tetracycline treatment did not affect the productivity of WM lines (unpublished results), but this may not hold for other stocks.

Levels of incompatibility in the field We have shown that the incompatibility between R males and W females seen in laboratory crosses is also occurring in a polymorphic natural population. In all three analyses of field-collected females from Piru, the egg- hatch for W females was significantly less than for R females. No significant difference was found in our single Lake Cachuma sample. We believe that our data from Piru are more reliable, because the collection site is a rural citrus grove in a predominantly agricultural area that harbors a dense D. simulans population. In contrast, our Lake Cachuma collection site is a heavily used campground with a low density

of flies, many of which may be recent immigrants. Hence, the critical random mating assumption of Equation 1 may not apply to our Lake Cachuma sample. Even at Piru, the level of incompatibility inferred from the field-collected females is much lower than expected from our laboratory experiments. Letting H denote the relative number of offspring produced by incompatible versus compatible crosses, our Piru estimates range from 0.52 to 0.81. Weighting these estimates by the inferred number of incompatible matings that contributed to them pro- duces a pooled estimate of H = 0.56 for our Piru samples. In contrast, for our laboratory experiments with R males of different ages (Figure 3), the relative egg hatch from incompatible crosses does not reach such high values unless the males are more than 2 weeks old.

Several hypotheses can explain this discrepancy. First, most matings in nature may involve fairly old males. This hypothesis is difficult to test, because of the difficulty of determining the age of wild-caught flies; but there is little evidence to support it and much indirect evidence against it. ROSEWELL and SHOR- ROCKS (1 987) estimated that cosmopolitan species of Drosophila, including D. simulans, live only a few days in woodland habitats. However, survival rates vary with habitat. For example, D. mercatorum adults from dry field sites are only a few days old, while adults from humid sites are mostly older than two weeks (JOHNSTON and TEMPLETON 1982). Nevertheless, laboratory studies of D. melanogaster (reviewed by PAR- TRIDGE 1988) suggest that male reproductive success reaches a maximum within the first week of adult life and then steadily declines. In addition, we found in our male-age experiments that hatch rates in control crosses decreased significantly with male age. The hatch rates observed for wild-collected R females (0.92-0.98) are comparable to the highest ones observed in our laboratory controls (0.89-0.91), suggesting that most matings in nature involve young males.

Second, females may mate assortatively i n nature and/or W males may have a systematic mating advan- tage, although we find no repeatable evidence for either phenomenon in the laboratory. Our average estimate of H from the Piru field-collected females is 0.56. To reconcile this with an H value of 0.42, corresponding to laboratory crosses with 1 2-day-old males, we require that W females in nature be more than 1.5 times as likely to mate with a W male as an K male. Assortative mating of this magnitude was unlikely to be detected in our cage experiments i n which roughly 100 mating pairs were examined. How- ever, reconciling the field estimate with H = 0 .3 , which corresponds roughly to our laboratory results with 6-day-old males, requires that W females be


roughly 2.25 times as likely to mate with a W male as an R male. Our cage experiments, which examined both relative mating success of R vs. W males and departures from random mating, would have detected such large departures from equal mating success and random mating with probability greater than 0.5. Thus, it seems unlikely that these factors are sufficient to account for the differences in our laboratory and field estimates of H . Testing for differential mating success in nature will require an assay for the incompatibility type of wild-collected males.

Third, R males in nature may generally harbor a lower level of infection than males in the laboratory and may therefore be more compatible with uninfected females. This hypothesis is indirectly supported by our finding that R females from nature occasionally produce W offspring, but R females in the lab do not. It could be directly tested with a quantitative assay for the level of infection.

Fourth, high temperatures in the field may suppress the incompatibility phenomenon. The temperatures at Piru routinely exceed 28 O during afternoons in the late spring, summer and early fall. HOFFMANN, TUR- ELLI and SIMMONS (1986) found that the incompatibility of R males and W females could be suppressed by rearing larvae at 28". We have not investigated the effects of daily temperature cycles with maxima above this critical value, but it seems plausible that such cycles could contribute to decreased incompatibility, possibly by reducing the level of infection. This temperature hypothesis can be tested by exposing a reference infected stock to field temperature conditions and checking levels of incompatibilty.

A fifth alternative is that W females in the polymorphic populations surveyed have evolved some resist- ance to the incompatibility system. Alleles that increase the compatibility of W females with R males would clearly be favored in a population that has remained polymorphic for many generations. Al- though HOFFMANN and TURELLI (1988) found no evidence for genetic variation among W females in their incompatibility with R males, intense natural selection for many generations will be much more effective in uncovering such variation than our laboratory tests. This evolutionary-response hypothesis can be tested by comparing the incompatibility levels shown by W females from locations with and without R males.

W offspring from R mothers: As noted above, our laboratory and field data give strikingly different results. In our laboratory experiments, R females have invariably produced R offspring, irrespective of their age or exposure to extreme temperatures. Despite extensive laboratory tests, we have previously re- covered both R and W progeny from only a single female who had inherited the infection paternally

(HOFFMANN and TURELLI 1988). We postulated that this female had received only a low level of the infection, which she did not transmit to all of her offspring. In contrast to this very rare event, we obtained both R and W sublines from 4 of 44 isofemale lines that were classified as type R when initially tested in the first laboratory generation from wild-caught females. Both R and W sublines were also obtained from another newly established isofemale line in the same collection that could not be assigned an incompatibility type. Moreover, two sublines initially characterized as W were characterized as R after additional generations of laboratory culture. These observations support the hypothesis that compatibility with our standard R males is a quantitative phenomenon rather than a qualitative one. A particular density of the microorganism may be necessary for complete compatibility and reliable maternal transmission. The data in BIN- NINGTON and HOFFMANN (1 989) suggest quantitative differences in infection levels between different R lines and between males of different ages. The increased compatibility in crosses with old R males is consistent with the lower infection level observed in these males. For some reason, after several generations of laboratory culture, R lines only produce R sublines. This may reflect the effects of selection in these cultures against females that do not carry a sufficient infection level to be compatible with their infected male sibs. Alternatively, conditions in nature may tend to suppress the infection. For instance, flies in some habitats may encounter antibiotics that partially cure the infection (STEVENS 1989). This is plausible because antibiotic-producing fungi are common on Drosophila resources.

Our Piru data provide a crude estimate of the fraction of W offspring from R mothers in nature. Ignoring the sublines from isofemale line 34 that could not be initially classified, three of the 430 sublines tested from R females were repeatably type W. Thus, we might expect at least 0.7% W offspring from R mothers. If there are many females in nature, like our line 34, that produce significant fractions of both R and W offspring and show an intermediate level of compatibility with R males, the estimate 0.7% may be far too low.

In our recent surveys, we have assayed isofemale lines as Fls from nature. In our 1989 Arvin collection, 22% of these lines could not be assigned an incompatibility type because of discrepancies between the out- comes of replicate crosses. Sublines from them are now being analyzed. We have previously noted unex- plained variability in the incompatibility reaction (6 HOFFMANN, TURELLI and SIMMONS 1986) and three isofemale lines that appeared to change incompatibility type (HOFFMANN and TURELLI 1988), but we have only recently begun the systematic study of sublines

946 A. A. Hoffmann, M . Turelli and L. G. Harshman

needed to document segregation of R and W offspring from individual field-collected females. Our current hatch-rate assay for incompatibility is much more discriminating that our earlier assay based on counting adult progeny production. In previous cases where ambiguities were noted, lines retested after additional generations of laboratory culture all gave unambiguous results. This is consistent with our findings that maternal transmission of R is stable in the laboratory and that no R sublines could be found from established W stocks (HOFFMANN and TURELLI 1988). Our new results indicate the importance of analyzing isofemale lines after a single generation in the laboratory. Analysis of sublines should be complemented by a quantitative assay for the incompatibility-causing microorganism in wild-caught flies.

Patterns in nature compared to theoretical predic- tions: In our initial description of this incompatibility system (HOFFMANN, TURELLI and SIMMONS 1986), we characterized California populations as either R or W. These designations were based on test crosses using mass cultures established from field-collected females several generations prior to testing. We found that the populations sampled south of the Tehachapi transverse range in southern California were R, while those to the north were W. This description was consistent with the simple theoretical model of CASPARI and WATSON (1959) which suggests that incompatibility types like R should either become fixed or lost in isolated populations. Our subsequent analyses of individual isofemale lines from more locations have revealed more complex patterns.

Persistence of a low frequency of W from predominantly R populations in southern Calqornia: All our samples of 12 or more isofemale lines from populations south of the Tehachapis contain a small but consistent fraction of W lines. The average frequency of type R in these populations is 94%. T o interpret this, we will ignore ambiguities in classification of wild-caught flies and consider some simple discrete-generation population models. We also assume that the incompatibility types are equally viable after egg hatch. It is possible that larvae from W females in polymorphic populations have higher viability or emerge as larger adults with higher fecundity than offspring of R females when eggs are laid on limited resources, because the reduced egg hatch may lead to lower competition among W larvae if they tend to compete among them- selves. SKINNER (1985) proposed this hypothesis to account for the persistence of a maternally inherited factor influencing sex-ratio in Nasonia. However, this scenario seems improbable for D. simulans. We expect that in polymorphic populations, R and W larvae generally co-occur, because more than one female will usually lay eggs on the same natural resource, as in D. melanogaster (HOFFMANN and NIELSEN 1985).

The easiest way to maintain uninfected individuals in a predominantly infected population is to have infected mothers produce some uninfected offspring (FINE 1978). T o quantify this, let H = 1 - sh denote the relative hatch rate from incompatible fertilizations (i .e. , fertilizations of W eggs by R sperm) let F = 1 - sf 1 denote the relative fecundity of R females, let p denote the fraction of offspring from R mothers that are W, and let P I denote the frequency of type R adults in generation t . Based on our experiments, we assume that sh > 0 and sf > 0. (Note that our model differs from FINE’S (1 978) which assumes that the infection equally affects the “fertility” of both males and females. Our experiments suggest that the infection affects female egg laying but not male mating success.) We will ignore the infrequent male transmission of the infection, because it has only a small effect on the values given below. Assuming random mating and equal viabilities for both incompatibility types, we have the recursions

Thus, the equilibrium frequencies for type R are p = 0 and the roots of

S h ( 1 - p + psf)p2 - (sf+ sh)P + sf+ p - pSf= 0. (3)

First note that if p = 0, corresponding to perfect maternal transmission, this equation yields two equi- libria: p = 1 and p = s f /$ , . Assuming that R females suffer a fecundity disadvantage (sf > 0), loss of the infection, i.e. p = 0, is always a locally stable equilibrium; if sh 5 sf, p = 0 is globally stable and the infection will be eliminated from an initially polymorphic population. If sh > sf> 0 with p = 0, then p = 0 and p = 1 are both locally stable; and the ultimate frequency of R is determined by whether its initial frequency is above or below the unstable equilibrium, p = s//sh (CASPARI and WATSON 1959). Our data suggest that sh > sf> 0, and reasonable estimates are sh = 1 - H = 0.3-0.45 and sf = 0.05-0.2.

With incomplete maternal transmission (p > 0) and sh > sf[ 1 - 2p(l - sf)], both roots of Equation 3 are between 0 and 1. The larger root is a stable equilibrium and the smaller is unstable. Assuming the frequency observed in southern California ( p = 0.94) is the stable polymorphic equilibrium, Equation 3 provides a simple relationship that must be satisfied by s h ,

sf and p. Using the extreme parameter combinations,

Equation 3 requires that p be between 0.008 and 0.039. Values up to p = 0.018 are consistent at the 5% level with our observed rate of segregation of W sublines from field-collected R females (the upper limit increases to p = 0.026 if we include the line 34 from Piru).

Sh = 0.3 and Sf = 0.2 US. Sh = 0.45 and Sf = 0.05,


An alternative explanation for the maintenance of a low frequency of type W is that these individuals are recent migrants from populations near fixation for type W. The most likely source of such individuals is central and northern California, where until recently all of our samples were type W. Recent analysis of mitochondrial DNA variation (A. A. HOFFMANN and S. W. MCKECHNIE, unpublished results) indicates that this is unlikely. The R and W types from Piru are differentially associated with mtDNA variants (HALE and HOFFMANN 1990): R flies all have the same hap- lotype (type B), whereas W flies have two haplotypes (A and B) with similar frequencies. W flies from central and northern California are predominantly type A, whereas R flies from the Riverside area are all type B. If “curing” is more important than migration in explaining the persistence of a low frequency of W near Riverside, then all of the rare W flies from Riverside should be type B. This was confirmed in a large collection from Riverside, suggesting that these flies arise from Riverside rather than from a distant W population.

Increase of R frequency in the Central Valley: Our September 1989 collection from Sanger was the first north of the Tehachapis to yield R isofemale lines, with the exception of two R lines collected in 1985 (HOFFMANN and TURELLI 1988). Because Sanger is on the eastern edge of the Central Valley approximately 120 miles north of the Tehachapis, we expected that the infection was spreading northward. The analysis of moving hybrid zones in BARTON (1979) can be adapted to show that if the unstable equilibrium p = sf/sh is below 0.5, type R is expected to spread through a uniformly distributed population at a rate governed by the selection coefficients and migration rates. To test the “wave of advance” hypothesis, we resampled Arvin in the southeast corner of the Central Valley, where the east-west transverse range (the Tehachapi) meets the north-south Sierra range. We expected that if the infection was spreading northward, this population would have a much higher frequency of R than Sanger. Instead we found that the frequency at Arvin was indistinguishable from that at Sanger (Table 3). This unexpected pattern may occur because the principal migration route is not north over the Tehachapis, but from coastal south-central California populations that have not been sampled.

Understanding the spatial and temporal pattern of spread will require describing on a finer spatial scale the transition from predominantly R to predominantly W populations. Estimates of the rate of spread of the infection could provide indirect estimates of average migration rates in natural fly populations, which are difficult to estimate directly (cf. COYNE, BRYANT and TURELLI 1987). The only inference that

can now be made from the observed increase to p 0.28 is that the unstable point p = sf/& must be below 0.28, so that sf< 0.28sh. Our field data suggest sh C 0.45, thus we expect the fecundity deficit for type R females to satisfy sf 5 0.13, which is consistent with our Piru data.

Persistent polymorphism at Piru: This polymorphism, documented for 2 years, is difficult to understand in terms of our experimental data. Twenty replicate laboratory populations initiated with 50% R and 50% W from Piru all approached virtual fixation for R within ten generations. Given the relatively mild cli- mate in this citrus-growing area, the Piru population must have produced at least ten generations in 2 years. One hypothesis to explain the polymorphism is that Piru represents a transition point between predominantly R and W populations, so that both types are maintained at high frequency by a balance between migration and within-population dynamics. However, as shown by BARTON (1979), stable contact zones between forms such as R and W tend to occur in areas of reduced population density. Our collection site is in the midst of a large agricultural area surrounded by relatively inhospitable habitat. Thus, it is likely to correspond to a peak of high population density rather than a trough.

Alternatively, the Piru population may be at a stable equilibrium maintained by a much higher rate of spontaneous curing ( i . e . , production of W offspring by R mothers) than we have documented. However, the conditions for a stable polymorphic equilibrium frequency of R at or below 0.7 seem quite restrictive. For instance, if sf = 0.1 and sh = 0.45, a stable equilibrium with p = 0.7 would require p = 0.092; but if this population were perturbed so that p < 0.632, R would be eliminated. Such an equilibrium is also very sensitive to increases in p. If p = 0.095 with sf= 0.1 and sh = 0.45, R will be eliminated irrespective of its initial frequency. If the fecundity deficit of R females is reduced and the hatch rate from incompatible crosses is increased, the conditions for a stable polymorphism at intermediate frequency are some- what relaxed. For instance, if sh = 0.35, sf= 0.05 and P = 0.074, the population approaches a stable equil- ilbrium frequency of R around 0.7 from all initial frequencies above 0.53. Such values of p may be reasonable given the large fraction of “ambiguous” females detected in our recent collection from Arvin. However, the very different polymorphisms in southern California and Piru can both be explained by this “mutation-selection balance” hypothesis only if the parameter values vary significantly between locations. This hypothesis can be tested by a more intensive investigation of newly established isofemale lines from different sites. Another possibility is that the selection and incompatibility parameters fluctuate in nature


(with temperature, for instance) so that R and W are favored in alternate seasons. Although this would not lead to a stable polymorphism, it would greatly retard the rate at which polymorphism is eliminated.

In conclusion, results from the laboratory have only been partially successful in explaining the distribution of R and W in the field. We are now at the stage where experiments need to be carried out in the field or with field-collected flies. This approach will be complemented by using molecular markers to meas- ure infection levels and to study the association between mtDNA variation and incompatibility. The R- W system represents an example of an endosymbiont with deleterious fitness effects that appears to be spreading. It provides an excellent opportunity to study the coevolution of hosts and their parasites.

We thank L. PARTRIDGE for helpful references, J. R. DAVID for sharing unpublished results, and A. G. CLARK, T. PROUT, J. H. WERREN and an anonymous reviewer for comments on earlier drafts of the manuscript that greatly improved the presention. We thank Z. CACOYIANNI, J. COFFER, S. O'DONNELL and K. PRAC for assist- ance in the laboratory, K. KERLE for computer programming, and B. RISKA for field collections. The hospitality of T. and C. KERSHAW of Sanger, California, and E. F. BURGER of Piru, California, is greatly appreciated. This work was partially supported by an Aus- tralian Research Committee grant to A.A.H. and National Science Foundation grant BSR-8866548 to M.T.

LITERATURE CITED

BARTON, N. H., 1979 The dynamics of hybrid zones. Heredity

BINNINGTON, K. C., and A. A. HOFFMANN, 1989 Wolbachia-like organisms and cytoplasmic incompatibility in Drosophila simulans. J. Invert. Pathol. 54: 344-352.

CASPARI, E., and G . S. WATSON, 1959 On the evolutionary importance of cytoplasmic sterility in mosquitoes. Evolution 13: 568-570.

COYNE, J. A,, S. H. BRYANT and M. TURELLI, 1987 Long-distance migration of Drosophila. 2. Presence in desolate sites and dispersal near a desert oasis. Am. Nat. 129: 847-861.

EFFRON, B., 1982 The Jackknge, the Bootstrap and Other Resampling Plans. Society for Industrial and Applied Mathematics, Phila- delphia.

FINE, P. E. M., 1978 On the dynamics of symbiote-dependent

43: 341-359.

cytoplasmic incompatibility in Culicine mosquitoes. J. Invert. Pathol. 30: 10-18.

GROMKO, M. H., M. A. NEWPORT and M. G. KORTIER, 1984 Sperm dependence of female receptivity to remating in Dro- sophila melanogaster. Evolution 3 8 1273-1282.

HALE, L. R., and A. A. HOFFMANN, 1990 Mitochondrial DNA polymorphism and cytoplasmic incompatibility in natural populations of Drosophila simulans. Evolution 44: 1383-1 386.

HOFFMANN, A. A., and K. M. NIELSEN, 1985 The effect of resource subdivision on genetic variation in Drosophila. Am. Nat. 125: 421-430.

HOFFMANN, A. A,, M. TURELLI and G. M . SIMMONS, 1986 Unidirectional incompatibility between populations of Drosoph- ila simulans. Evolution 4 0 692-701.

HOFFMANN, A. A,, and M. TURELLI, 1988 Unidirectional incompatibility in Drosophila simulans: inheritance, geographic variation and fitness effects. Genetics 119: 435-444.

JOHNSTON, J. S., and A. R. TEMPLETON, 1982 Dispersal and clines in Opuntia breeding Drosophila mercatorum and D. hydei at Kamuela, Hawaii, pp. 241-256 in Ecologzcal Genetics and Evo- lution: The Cactus-Yeast-Drosophila Model System, edited by J. S. F. BARKER and W. T . STARMER. Academic Press, Sydney.

KARLIN, S., and J. MCGREGOR, 1972 Applications of method of small parameters to multi-niche population genetic models. Theor. Popul. Biol. 3: 186-209.

Lours, C., and L. NIGRO, 1989 Ultrastructural evidence of Wol- bachia Rickettsiales in Drosophila simulans and their relationship with unidirectional cross-incompatibility. J. Invert. Pathol. 54: 34-44.

NIGRO, L., and T. PROUT, 1990 Is there selection on RFLP differences in mitochondrial DNA? Genetics 125: 551-555.

PARTRIDGE, L., 1988 Lifetime reproductive success in Drosophila, pp. 11-23 in Reproductive Success, edited by T. H. CLUTTON- BROCK. University of Chicago Press, Chicago.

PYLE, D. W., and M. H. GROMKO, 1981 Genetic basis for repeated mating in Drosophila melanogaster. Am. Nat. 117: 133-146.

ROSEWELL, J., and B. SHORROCKS, 1987 The implication of survival rates in natural populations of Drosophila: capture-recap- ture experiments on domestic species. Biol. J. Linn. SOC. 32:

SKINNER, S. W., 1985 Son-killer: a third extrachromosomal factor affecting the sex ratio in the parasitic wasp, Nasonia (=Mormon- iella) vitripennis. Genetics 109: 745-759.

SOKAL, R. R., and F. J. ROHLF, 1981 Biometry, Ed. 2. Freeman, New York.

STEVENS, L., 1989 Environmental factors affecting reproductive incompatibility in flour beetles, genus Tribolium. J. Invert. Pathol. 53: 78-84.

373-384.

Communicating editor: A. G. CLARK

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