tyes, AH162, collected in Apple Hill, near Davis, CA, in 1982 and BogER ..... genetic reorganization in a small founder population. .... Alan R. Liss, Inc., New York.
Copyright 0 1996 by the Genetics Society of America
Cytoplasmic Incompatibility and Mating Preference in Colombian Drosophila pseudoobscura Tracie M. Jenkins,’ Christina S. Babcock,* DavidM. Geiser’ and Wyatt W. Anderson Department of Genetics, University of Georgia, Athens, Georgia 30602 Manuscript received September 15, 1994 Accepted for publication October 4, 1995 ABSTRACT and ANDERSON observed a large frequency change of mitochondrial DNA (mtDNA) haplotypes in a population initiated with two allopatric strains of Drosophila pseudoobscura, BogER from Colombia and AH162 from California. They concluded that mtDNA haplotypes in D. pseudoobscura are not always selectively neutral. NIGROand PROUTsuggested, however, thata maternally transmitted incompatibility system, similar to the one they observed in two strains of D.simulans from Italy, could account for the observed mtDNAfrequency changes. SINCHand HALE postulated that a mating preference between the strains BogERand AH162 in MACRAE andhDERSON’sexperiment, in the formof negative assortative mating, could also account for the mtDNA frequency changes. We report two experiments designed to test the hypotheses: that a maternally transmitted cytoplasmic incompatibility system exists between D. pseudoobscura strains BogERand AH162; and, that BogER females mate preferentiallywith AH162 males. Our results do not support either hypothesis. MACRAE
M
and ANDERSON (1988) set up experiments to test the hypothesis that mitochondrial DNA (mtDNA) is a selectively neutral marker. They used Drcsophila pseudoobscura strains with unique mtDNA haplotyes, AH162, collected in Apple Hill, near Davis, CA, in 1982 and BogER, collected in 1978 near Bogota, Colombia. Male hybrids with Colombian mothers and California fathers are sterile while malesfrom the reciprocal cross and females from either cross are fertile. BogER mtDNAfrequency increased 46% overjust three generations in their first population cage. This result, like those of CLARK and LYCKEGAARD (1988) with different geographic strains of D. melanoguster, AUBERTand SOLIGNAC(1990) with D. simulans and D. mauritiana and Fos et al. (1990) with different geographic strains of D. subobscura, suggests that mtDNA is not always a selectively neutral marker. It has been proffered, however, that other explanations such as a cytoplasmic incompatibility (NIGROand PROUT1990; KAMBHAMPATI et al. 1992) or mating preference (SINGHand HALE 1990) are more plausible for the MACand ANDERSON (1988) results than nonneutrality of the mtDNA marker. NIGROand PROUT(1990) used strains of D. simulans and KAMBHAMPATI et al. (1992) used strains of Aedes albopictus to show that mtDNA frequency changes in AcRAE
Corresponding author: Wyatt W. Anderson, Arts and Sciences, 310 New College, University of Georgia, Athens, Georgia 30602-1732. Present address: Basic Sciences, Department of Genetics, Mercer University School of Medicine, Macon, GA 31207. * Present address: Department of Ecology and Evolution, University of Arizona, Tucson, AZ 85721. Present address: Department of Plant Biology, University of California, Berkeley, CA 94720.
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Genetics 142 189-194 (January, 1996)
cage populations could be explained by an intraspecific reproductive incompatibility (TURELLI et al. 1992). Studies also suggested (POWELL 1982), butdid not demonstrate, amicrobial symbiont in D. pseudoobscura (J.R. POWELL,personal communication). MAcRAEand ANDERSON (1988) subjected the FJ1 generation of the AH162/BogER cage to both tetracycline treatment and heat shock, which hadbeen shown to be effective against microorganisms responsible for cytoplasmic incompatibility in Drosophila (HOFFMAN et al. 1986). They observed no effect from these treatments and concluded that a cytoplasmic microorganism was not responsible for the changes in haplotype frequency (MACRAE and ANDERSON 1990). NIGRO and PROUT(1990) and KAMBHAMPATI et al. (1992), however, maintain that a cytoplasmic incompatibility (CI) caused by a Wolbachia rickettsia and unidirectionally inherited through the egg cytoplasm, rather than selection on mtDNA, can adequately explain mtDNA frequency changes in the MAcRAE and ANDERSON (1988) study. PRAKASH (1972) and SINGH(1983) both studied the mating behavior of D. pseudoobscura collected in Bogota by ALICE HUNTERin 1966 (PRAKASH and LEWONTIN 1968) but produced different results. PRAKASH (1972) found that female flies from Bogota mated randomly with D. pseudoobscura males fromthe United States. SINGH(1983), however, in an extensive study of isofemale lines from Bogota and the United States, demonstrated a patternof negative assortative mating in which Bogota females preferentially mated with males from the United States. Based on these latter results, SINGH and HALE (1990) proposed that mating preference was a more likely explanation for the mtDNA frequency changes observed by MAcRAE and ANDERSON (1988)
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than was selection on mtDNA haplotypes. SINCHand HALEcontend that BogER females, although mated (EVIAcRAE and ANDERSON 1990) before being put into population cages, continued to mate more often than AH162 females and particularly more often AH162 with males. MAcRAE a n d ANDEMON (1990) argued against this line of reasoning on t h e basis that it is the female, not the male, that transmits mtDNA. SINCHand HALE (1990) contend, however, that the mating behavior of t h e BogER female would result ina higher starting frequency for the BogER mtDNA haplotype and in addition would lead to a higher frequency of pure BogER flies in the F, generation. This situation would perpetuate the mating bias of the BogER parental females, as well as favor theBogER haplotype overthe AH162 haplotype. paper is to r e p o r t o n two experiThe purpose of this ments designed to determine if either a GI system or negative assortative mating can account for ~ the ~Ac:RAE a n d ANDEMON (1988) results. First, we measuredviability (egg-to-adult survival) to test the hypothesis that a cytoplasmic incompatibility system caused a sizable red u c t i o n i n t h e n u m b e r o fF1 adult progeny in matings between BogER females andAH162 males. Second, we used salivary chromosomes and mtDNA haplotypes to determine the mating pattern in a population formed by mixing the BogER a n d AH162 strains, to test the hypothesis that BogER females from Colombia prefer to mate with AH162 males from California. MATERIALSAND METHODS Viability experiment, a test for cytoplasmic incompatibility: Viability is defined as survival ofF, progeny from the eggto-adult life history stages. Reduced viability of F, progeny with parents from different geographical populationsis indicative of a maternally inherited microorganism or cytoplasmic incompatibility system (HOFFMAN et al. 1986; HOFFMANand 1988; NIGRO1991). Our experimental design thereTURELLI fore utilized viability as the index for cytoplasmic incompatibility. The purity of BogER and AH162 strains was first confirmed from salivary chromosome preparations (ANDERSON et al. 1979). The strains were then raised on standard cornmeal medium (ANDERSON et al. 1986),either with 0.3% tetracycline to remove tetracycline-sensitive cytoplasmically transmitted microorganisms such as Wolbachia (HOFFMAN et al. 1986) or without tetracycline, for two generations prior to being mated. The rest of the experiment, including all matings, was conducted on the type of media on which the flies were raised for two generations previously. Thus all crosses were done both on media with tetracycline and media without tetracycline. Initially, five replicates were set up foregg-laying chambers (ANDERSON et al. 1986) in each of the four female X male crosses (BogER X BogER, AH162 X AH162, BogER X AH162 and AH162 X BogER). Each cross was made with 20 virgin females and 20 virgin males, 3-7 days old. The flies were allowed to mate in the laying chambers at 24" in the dark until the females began to lay eggs. Because some of the initial eggs laid by virgin females may be unfertilized, eggs from the first day of laying were discarded. Both females and males were then transferred to new egg-laying
chambers for at least four more hours. Batches of 25 eggs were transferred from theegg-laying chambers to 8-dram glass vials filled with 7 ml of the same medium as in the egg-laying chamber. Eighteen hundred eggs in total were divided equally among 72 glass vials per cross (36 with and 36 without tetracycline) and incubatedat 24" in the dark. Adult progeny began to emerge in -2 weeks, and they were counted twice each day until no adult progeny emerged for three consecutive days. The results were recorded by sex, aswellasby total number of survivingadults. Viability was measured as the average number of F, adult progeny to survive over all vials per type of medium. Mating preference experiment, a test for negativeassortative mating: The design of this experiment relied on two facts. First, AH162 is homokaryotypic for the Standard (ST) third chromosome inversion type, and BogERis homokaryotypic for TreeLine (TL). Second,mtDNA is only inherited through the mother. Two thousand adult flies made up of equal numbers of each sex and strain were added to a population cage and allowed to mate. The females were virgins and all of the adults were between 3 and 7 days old. After 3 days of mating, 436 females were vacuumed fromthe cage and placed individually into 8-dram glass vials to lay eggs. Our goal was to determine the patternof matings for 2200 females. All 436 females mated and produced offspring, and from them we selected a random sample of 230 females for further study. One larval offspring from each female was scored for its inversion type (ANDERSON et al. 1979). When a larva was homokaryotypic, its parents were easy to identify as being the same homokaryotype as the larva. When an offspring larva was heterozygous for ST and TL, however, it was not possible to determine from salivary chromosomes alone which genotype was the mother and which was the father. We therefore used the difference in mtDNA haplotypes of the two strains to establish maternity. Ninety-five of the 230 F1 offspring whose parents we wanted to deduce were heterokaryotypes. The maternal genotypes of these heterozygous offspring were determined from restriction digests of their sibs, with whom they shared a common mtDNA haplotype. The restriction fragments of MspI digests and ANDERSON were characterized for bothstrains by MAC(1988). Following their technique, DNA from single adult flies was extracted, and after digestion with MspI, it was probed with "P-labeled mtDNA fragments from D. pseudoobscura. RESULTS
Viability experiment, a test for cytoplasmic incompatibility: An analysis of variance indicated no effect of tetracycline on m e a n progeny viability ( P = 0.45). The test cross made in media without tetracycline should have been particularly susceptible to a cytoplasmic innot be expected if a compatibility. This result would cell-wall deficient microorganism causing cytoplasmic incompatibility, like a Wolbachia rickettsia, were present in the cytoplasm.The viability data for each of the four crosses (Table 1) were therefore collapsed into a single quantity summarizingsurvival in 72 replicate vials with 25 eggs per vial a n d 1800 eggs total. Viabilities and numbers of adults were compared between crosses and between sexes by means of two-sample t-tests. A comparison of femaleand male survival ( P = 0.15) revealed no difference between the sexes.The progeny from the BogER X BogER (B X B) parental control cross had the highest viability ofallcrosses,
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TABLE 1 Vlabfity of eggs produced by four parental crosses
Cross“ (female Male X male)Female
Total ~~~~
No. of adult progeny
Percent viability
~
B A B A
X X X X
B A A B
91.9 90.8 88.6 87.7
? 2 rfr rfr
0.07 0.08 0.10 0.11
11.82 2 0.27 11.54 f 0.27 11.07 2 0.28 11.26 ? 0.30
11.15 f 0.27 11.54 ? 0.27 11.10 f 0.25 10.67 2 0.31
22.97 22.63 22.17 21.93
2 0.21
f 0.19 t- 0.19 rfr 0.22
Values are means 2 SE. Viability measured as percent of eggs that developed into adults and as mean number of eggs per vial that developed into adults. B, BogER strain and A, AH162 strain.
91.9%, although notsignificantly more ( P = 0.20) than viability in the parental control cross AH162 X AH162 (A X A), which was 90.8%. The cross that produced fertile hybrid males, AH162 X BogER (A X B), had the lowest viability of allcrosses, 87.796, while the cross that produced sterile hybrid males, BogER X AH162 (B X A), had a viability of 88.6%. There was no significant difference in viability between the two hybrid crosses ( P = 0.10). Likewise, the numbers of adult females from the B X A and A X B crosses ( P = 0.25) were not significantly different. But the same comparison of the number of adult males ( P = 0.03) indicated that significantly more adult males developed from the B X A cross than the A X B cross (Table 1). Thus, while B X A hybrid males are sterile, they do notshow the inviability that is expected with cytoplasmic incompatibility between the strains being crossed. Mating preference experiment, a test for negative assortative mating: The parents of 230 randomly selected progeny were determined from salivary preparations: 89 ST/ST progeny produced by A X A matings; 46 TL/TL progeny produced by B X B matings; and 95 ST/TL progeny produced by A X B or B X A matings. Southern blot analysis of mtDNA restriction digest revealed that 34 of the 95 heterokaryotypes had B mothers and 54 had A mothers, while the maternity of seven heterokaryotypes could not be determined for technical reasons. The relative frequencies of each of the four kinds of matings in our population cage were compared with the expectations under random mating by means of chi-square tests for goodness of fit.The relative frequencies of females in our sample and the relative frequencies of the males who mated them were multiplied to generate the expected frequencies of matings. The 223 mating pairs established from salivary preparations and mtDNA haplotypes are presented in Table 2, along with the statistical analysis. Although we expected to collect an approximately equal number of AH162 and BogER females, 64% of the females were from strain AH162. The mtDNA haplotype carried by AH162 females, however, is not the
TABLE 2 Frequency of matings between females and males from BogER ( B ) and AH162 (A) strains No. of offspring karyotypes
Homozygotes Heterozygotes
x*
B X BA X AB X AA X B
Observed number” Adjusted number* Homozygotes reduced Expected number BogER mothers assumed Expected number AH162 mothers assumed Expected number
46
89
34
54
42.6 34.7
82.4 74.6
34.0 41.9
54.0 61.9
5.1*
46.0 37.8
89.0 80.8
41.0 49.2
54.0 62.2
5.1*
46.0 37.2
89.0 80.2
34.0 42.8
61.0 69.8
6.0*
The fit to random mating was tested by chi-square, d.f. = 1. Asteriskindicatesfailureto fit at 0.05 probabilitylevel. Frequencydeterminedfromnuclearandmitochondrial markers and tests for fit to random mating. a Genotypes determined from nuclear and mitochondrial for whichmaternity markers,minussevenheterozygotes could not be determined. B, BogER and A, AH162; B X A = B female X A male. Numbers adjusted to take accountof the seven “undetermined” heterozygotes. haplotype that dramatically increased in the MACRAE and ANDERSON study (1988).The difference in the frequencies of AH162 and BogER females in our sample was statistically significant (x2 = 17.8, d.f. = 1, P < 0.01). Slightly more than halfof the matings (55%) involved AH162 males, but this difference was not significant (x2= 1.8, d.f. = 1, P = 0.18). We were unable to determine the maternity of seven of the heterokaryotypes and therefore had to compensate for them in our analysis of fit to random mating. First, we took the conservative approach of reducing the homozygotic frequencies by the proportion of heterozygotes whose mtDNAhaplotype could not be determined, 7/95 (Table 2). We believe that the resulting
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frequencies of males and females mating in the cage are the best estimates from our data. BogER females mated withBogERmales more often than expected under random mating, and AH162 females mated with AH162 males more often than expected, while BogER females mated less often with AH162 males than expected and AH162 females mated less often with BogER males than expected. Thus there was positive assortative mating in the population, and the departure from random mating was statistically significant ( x 2 = 5.1, d.f. = 1, P = 0.02). Second, we assumed that the seven heterokaryotypes for which maternity could not be determined all had BogER mothers(Table 2). The chi-square value remained significant (x' = 5.1, d.f. = 1, P = 0.02) and continued to indicate positive assortative mating. Third, we assumed that the seven heterozygotic offspring forwhich maternity could not be determined all had AH162 mothers (Table 2). The chi-square value (x' = 6.0, d.f. = 1, P = 0.01) was significant, and again females of each strain preferred to mate with males of their own strain more often than expected and with males of the different strain less often than expected. Clearly, our results show that BogER females prefer BogER males over AH162 males. DISCUSSION
Like J. R. POWELL(personal communication), who designed experimentsto test the existence of a microorganism in D.pseudoobscuru beginning in 1979, we find no evidence for a tetracycline-sensitive endosymbiont. We conclude that a cytoplasmic incompatibility is not responsible for the hybrid male sterility observed when BogER females are mated to AH162 males. The large numbers of progeny studied, 18,000 eggs per cross, permitted us a robust analysis of the viability of F1 progeny, testing for the presence of the cell-wall deficient microorganism typical of cytoplasmic incompatibility. There was no difference between the amount of adult progeny produced on media with tetracycline compared with media without tetracycline. We conclude that, although atetracycline-sensitive endosymbiont has been shown to be the cause of reduced viability in Drosophila (TURELLI and HOFFMAN 1991; HUTTER and RAND 1995), there is no cell-wall deficient microorganism mediating CI between strains BogER and AH162. A mycoplasma-like organism, which is sensitive to tetracycline, has been implicated in unidirectional hybrid sterility in Drosophila (GOTTLIEB et ul. 1981). However, unidirectional hybrid male sterility in Drosophila generally (PEREZet al. 1993; ZENC and SINGH1993), and for D. pseudoobscuru specifically (ORR 1989), has been shown to be genetic in nature with the Xchromosome exerting the most effect. F, hybrid male sterility that results when BogER females mate AH162 males maps
largely to the X chromosome (COYNEand ORR1986; ORR1989). If this hybrid male sterility was the result of a cell-wall deficient endosymbiont, F1 hybrid male progeny raised on media with tetracycline should have demonstrateda significant increase in fertility. Our study clearly shows that hybrid male sterility was independent of the type of media on which the flies were raised. Furthermore, our data showed that hybrid male progeny with BogER mothers, although sterile, are as viable and as numerous as their female siblings. They are even significantly more viable than males from thereciprocal cross. The cross that produced theleast viable progeny, the one that produces fertile hybrid males (AH162 X BogER), still produced -88% viable progeny. There was no significant reduction in egg-to-adult viability of hybrid progeny with BogER mothers over hybrid progeny with AH162 mothers, as would be expected in a cytoplasmic incompatibility system (HALE and HOFFMAN 1990). We reject the hypothesis, therefore, that a cytoplasmic incompatibility occurs in crosses between D. pseudoobscuru strains BogER and AH162. The dramatic change in mtDNA frequencies observed by MACRAE and ANDERSON (1988) cannot be attributed to a cytoplasmic incompatibility as demonstrated by NICRO and PROUT(1990) and KAMBHAMPATI et ul. (1992). MACand ANDERSON (1988) suggest that the dramatic mtDNA frequency changes that they observed may be due to mtDNAs interacting with nuclear genes. The initial disequilibrium established between a mtDNA haplotype and a nuclear gene or genes could produce significant mtDNA changes. As the disequilibrium decayed, however, the mtDNA frequency changes would be reduced. Fos et. a1 (1990) demonstrated significant mtDNA haplotype frequency changes on different nuclear backgrounds. Their findings suggest that the nonneutral behavior of mtDNA haplotypes in their experiments may be due to cytonuclear interactions. HUTTERand RAND (1995) showed a significant fitness advantage for D.pseudoobscuru nuclear fertility factors and D. pseudoobscuru mtDNA on its own genetic background, a slight but nonsignificant fitness advantage of D. pseudoobscuru mtDNA on D.persirnilis nuclear background and nosignificant advantage in reciprocal test. These results provide some evidence that D.pseudoobscUTU nuclear and mitochondrial genomes are part of a coadapted genetic complex, and as such, may be subject to selection. The pattern of mating between BogER and AH162 strains ina large experimentalpopulation was estimated by determining the parentage of progeny that resulted from these matings through the examination of chromosomal and mtDNA markers. Although randomly swept from a population cage begun a few days earlier with equal numbers of AH162 and BogER females, significantly more AH162 females were collected than BogER females. It is not clear to us why the two
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hypotheses for mtDNA haplotype frequency changes, strains were unequally represented in our sample of the possibilityof genetic hitchhiking of the mtDNA females from the cage. The haplotype carried by the onto a nuclearbackground where loci are either under AH162 female, however, did not increase dramatically selection or in linkage disequilibrium with other loci in the MACRAE and ANDERSON (1988) study. This haplothat areunder selection cannot bediscounted. MACRAE type constituted 70% of the first cage populations in which the BogER haplotype increased dramatically. and ANDERSON (1988) observed that when Cage 1 was The mating pattern in this study was consistent with a perturbedthere was an immediate drop in BogER slight tendency to positive assortative mating, and inmtDNA frequency. However, the BogERmtDNA freconsistent with negative assortative mating. quency returned to the equilibrium frequency existing If negative assortative mating were the mechanism before perturbation. Could this phenomenon be the that caused the 46%mtDNA haplotype frequency result of the mtDNA hitchhiking on a nuclear backchange observed in the MAcRAE and ANDERSON (1988) ground in which a gene in linkage disequilibrium with other loci is undergoing balancing selection (HEDRICK study as suggested, then it should also be a detectable 1983)? It would be interesting to theoretically determechanism in our experiment. Our data, however, supmine if nonneutrality of mtDNA haplotypes could be port the expectation of DOBZHANSKY and MAYR (1944) the result of nuclear-mitochondrial interactions, and that strains of Drosophila will generally show positive assortative mating, which in thiscasewouldserve to how much selection on nuclear genes would be needed reduce the numbers of sterile male hybrids. We thereto produce the mtDNA frequency changes observed by MACRAEand ANDERSON (1988) under a hitchhiking fore reject the hypothesis that BogER females mate preferentially with AH162 males. Negative assortative effect. mating cannot account for the mtDNA haplotype freRAND et. al (1994) demonstrated that analyses of sequency changes observed by MAcRAE and ANDERSON quence data from the mitochondrial gene ND5 in two species of Drosophila generally supported a neutral (1988). model. The RAND et. al (1994) analyses also provided SINGH(1983) studied strains of D. pseudoobscura from Colombia collected in 1966 by ALICE HUNTER, some evidence for nonneutral evolution in selected rewhereas the BogER strain used by MAcRAE and ANDERSON gions of this gene. Evidence continues to mount increand SOLIG (1988) was collected in 1978 by HUGO HOENIGSBERG. mentally for selection on mtDNA (AUBERT Like SINCH(1983), we observed genetic variability in NAC 1990; FOS et al. 1990; RAND et al. 1994). Nonneutral fitness characters among hybrid F1 progeny resulting behavior of mtDNA should be considered, therefore, from allpossiblecrosses between seven Colombian as a possiblility, particularily when other factors such as strains (includingthe BogER strain) and a United cytoplasmic incompatibility and mating choice are States strain in terms of development time, number of ruled out. progeny and sex ratio (T. M. JENKINS, unpublished We thank MICHAELARNOLD, MARJORIE ASMUSSEN, JOHNAVISE, SIDdata). This variability could account for the difference NEY KUSHER,A M Y M A C U ,TIMPROUT,RAMA SINC:H and MICWI. in mating behavior of the BogER females from that of TUREI.I.I for their comments on this manuscript. We are grateful to ROBERTSOW. for his advice on the statistical analysis of the mating the older Colombian strains used by SINGH (1983). preference experiment, andto HOSSAINAIAVI for his expert technical Our data show that the dramatic BogER mtDNA freassistance. quency change observed by MAcRAE and ANDERSON (1988) cannot be attributed to either cytoplasmic inLITERATURE CITED compatibility or to negative assortative mating. The genetic differentiation in Colombia has led to partial hyANDERSON, W. W., J. ARNOLD, S. A. SAMMONS and D. G. YARDLEY, brid sterility, butnot to cytoplasmic incompatibility. 1986 Frequencydependent viabilities of Drosophila pseudoobscura karyotypes. Heredity 56: 7- 17. Also, the mating pattern may reflect on the size of the ANDERSON, W. W., L. LEVINE, 0. OLVERA, J. R. POWELL, M. E. DE LA Colombian population as well asthe stage of speciation. 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