Copyright Q 1996 by the Genetics Society of America

A Genome-Wide Survey of Hybrid Incompatibility Factors by the Introgression of Marked Segments of Drosophila mauritiana Chromosomes into Drosophila simulans

John R. True,* Bruce S. Weirt and Cathy C. Laurie”

*Department of Zoology, Duke University, Durham, North Carolina 27708 and tDepartrnent of Statistics, North Carolina State University, Raleigh, North Carolina 27695 Manuscript received August 31, 1995 Accepted for publication November 29, 1995

ABSTRACT In hybrids between Drosophila simulans and D. mauritiana, males are sterile and females are fertile, in compliance with HALDANE’S rule. The geneticbasis of this phenomenon was investigated by introgression of segments of the mauritiana genome into a simulans background. A total of 87 positions throughout the mauritiana genome were marked with P‘lement insertions and replicate introgressionswere made by repeated backcrossing to simulans for 15 generations. The fraction of hemizgyous X chromosomal introgressions that aremale sterile is -50% greater than the fractionof homozygous autosomal segments. This result suggests that male sterility factors have evolvedat a higher rate on the X, but chromosomal differences in segment length cannot be ruled out. The fractionof homozygous autosomal introgressions that are male sterile is several times greater than the fraction that are either female sterile or inviable. This observation strongly indicates that male sterility factors have evolved more rapidly than either female sterility or inviability factors. These results, combined with previous work on these and other species, suggest that HALDANE’S rule has at least two causes: recessivity of incompatibility factors and differential accumulation of sterility factors affecting males and females.

NE of the most general and striking patterns in hybrids have different genotypes (due to heteroga- 0 evolutionary biology is known as HALDANE’S rule, mety) or whether it reflects a difference in the rates which describes the results of interspecific crosses: of accumulation of male us. female incompatibility whenever hybrids of just onesex are inviable or sterile, factors. it is nearly always the heterogametic sex (HALDANE 3. In Drosophila and mammals, there are many more 1922). This rule applies very well to a great diversity of hybridizations that follow HALDANE’S rule forsterility different organisms, including insects and mammals (in thanfor inviability (Wu and DAVIS 1993), which which males are heterogametic) aswell as birds and raises the question of whether male sterility factors lepidopterans(in which females areheterogametic) accumulate morerapidly during evolution than invi- (COWE and ORR1989a; WU and DAVIS1993). In recent ability factors. years, several other patterns have been detected in the 4. In lepidopterans and birds, the numberof hybridiza- descriptive data onhybrid incompatibilities, which have tions that follow HALDANE’S rule for sterility (36 and stimulated theoretical work and raised important ques- 21, respectively) is similar to the number that follow tions about the evolution of postzygoticisolating mech- the rule for inviability (15 and 30, respectively) (WU anisms: and DAVIS 1993). This observation, in contrast to that in the preceding paragraph, raises the question In Drosophila, reciprocal crosses often producester- of whether the relative rates of evolution of male ile males in one direction, but not the other (BOCK sterility, female sterility and inviability factors differ 1984).This asymmetryis expected under simple between organisms with heterogametic males or fe- models for theevolution of genetic incompatibilities males. (Wu and BECKENBACH1983; TURELLIand ORR 5. There appear to be more exceptions to HALDANE’S 1995). rule for inviability in Drosophila than in birds or In Drosophila, hybridizations in which females are lepidopterans (WUand DAVIS1993). One hypothesis sterile involve more distantly related species than to explain this effect is that maternal us. zygotic in- those in which only males are sterile (COYNEand compatibilities are expected to occurmore fre- ORR1989b). An important question is whether this quently when the motheris homogametic than when observation is due to the fact that male and female she is heterogametic (Wu and DAVIS 1993). Excep- tions to HALDANE’S rule may also depend on the Curresponding author: Cathy C. Laurie, DCMB/Zoology, Box 91000, Duke University, Durham, NC 27708. number of incompatibility factors and their degree E-mail: [email protected] of dominance (TURELLIand ORR1995).

Genetics 142 819-837 (March, 1996) 820 J. R. True, B. S. Weir and C. C. Laurie

It is clear that resolution of the issues raised by the specific gene interactions are particularly deleterious descriptive data on hybridization patterns can be re- when at least one member of the gene pair is hemizy- solvedonly by careful genetic analyses. Fortunately, gous or homozygous for species A and the othermem- because at least one sex is viable and fertile in many ber is heterozygous or homozygous for species B. interspecific crosses, the genetic basis of hybrid incom- Recently, MULLER’Sideas about heterospecific gene patibility is amenable to direct experimental analysis interactions have been formalized and expanded into and many such studies have been done with Drosophila a “dominance theory” of HALDANE’Srule (Om 1993a; species in the past several years (see COYNEand Om TURELLIand Om 1995). This theory explicitly deals 1989a; COYNE 1992;WU and DAVIS1993; Wu and PALO- with an important factor that was not considered by POLI 1994). MULLER,which is that hybrid females with two Xchro- The most common experimental design in genetic mosomes have, on the average, twice as many potential studies of male hybrid sterility in Drosophila is the stan- X-autosome incompatibilities as XY hybrid males. dard backcrossanalysis. In this design, females with Therefore, if heterozygous gene pairs have some degree markers on each chromosome arecrossed to unmarked of deleterious interaction, it is possible for hybrid fe- males of another species, the heterozygous hybrid fe- males to have a lower fitness than hybrid males even if males are backcrossed to males of the multiply marked hemizygous-heterozygous interactions are worse than species, and progeny genotypes are analyzed for differ- heterozygous-heterozygous ones. The model of hybrid ences in sterility level. Several different pairs of Dro- fitness developed by TURELLIand Om (1995) shows sophila species have been analyzed in this way (reviewed that X-linked incompatibility factors have greater cumu- by COYNEand ORR 1989a).Segregation of the X chro- lative effects in the hemizygous XY male than in the mosome marker typically has a much larger effect on heterozygous XXfemale onlyif they are partially or fully male fertility than segregation of any of the autosomal recessive. markers, although each autosomal marker usually has A potentially powerful test of the X-autosome imbal- a significant effect. In some cases the Y chromosome anceand dominance theories is to comparethe fit- plays an importantrole (VIGNEAULTand ZOUROS1986), nesses ofhybrid males and females that have equivalent while in others it has no apparent effect (JOHNSON et genotypes in terms ofX-autosome imbalance. COYNE al. 1992; ZEN(; and SINCH1993). (1985) used an attached-X stock of D. simulans to pro- The causes of the large effect of the Xchromosome duce unbalanced hybrid females that are homozygous in backcross analyses withDrosophila species has gener- for the simulans X and heterozygous for simulans and ated much discussion in the literature, but the issue is maum‘tiana (or sechellia) autosomes. In crosses to both still not resolved. One possibility is that the Xcontains maum‘liana and sechellia, the unbalanced females are fer- a disproportionate number of sterility factors, which tile, whereas the genotypically similar males (hemizy- may be due to a higher Xchromosomesubstitution rate gous for the simulans X and heterozygous for the au- in the pure species populations (CHARLESWORTHet al. tosomes) are sterile. COYNEargued that if ordinary 1987; COYNEand Om 1989a). Anotherpossibility is that hybrid females are fertile (while males are sterile) be- the large Xeffect is due to dominance because, in the cause of the recessiveness of X-linked incompatibility backcross design, hemizygous X effects are compared factors, then the unbalanced females should be sterile. with heterozygous autosomal effects (COYNE and Om However, this interpretation is complicated by the fact 1989a; Wu and PALOPOLI1994). If the autosomal genes that male and female fertility are controlled by largely that cause hybrid sterility are not fully dominant, the different sets of genes (LINDSLEYand TOKUYASU1980), hemizygous X chromosome will have a greater effect which mayevolve at different rates (Om 199313; WU than a heterozygous autosome, even though it may con- and DAVIS1993). Thesimulans Xchromosome may not tain the same number and type of sterility factors. contain incompatibility factors that arecapable of caus- The relative effects of hemizygous and heterozygous ing hybrid female sterility even when homozygous. chromosomes in hybrids is a central issue in the X- Therefore, these results do not provide a strong argu- autosome imbalance hypothesis proposed by MULLER ment against the notion that sterility factors are gener- (1940) to explain HALDANE’S rule. Homogametic hy- ally recessive. brids have one complete set of chromosomes from each Similar comparisons of unbalanced male and female species whereas heterogametic hybrids have a set of genotypes have been made for two Drosophila species autosomes from each species, but only one X chromo- pairs that follow HALDANE’Srule with respect to inviabil- some. MULLERsuggested that this X-autosome imbal- ity (Om 1993b). In this case, unbalanced females are ance in heterogametichybrids can result in “disharmo- inviable, in contrast to the fertility results. The most nies in functioning”, leading to inviability or sterility. likely explanationfor this difference is that viability He also recognized that similar incompatibilities may genes typically affect both sexes in asimilar way whereas result in F2 hybrid breakdown when some parts of the fertility genes are generally sex specific. These results autosomes become homozygous while others remain are consistent with the dominance theory. Ordinary hy- heterozygous. In effect, the hypothesis is that hetero- brid females with an Xfrom each species are expected Hyhrid IllCOmpdtibility in Drosophila 82 I to contain twice as many X-linked inviability factors as D. mauritiana - the attached-X hybrid females,vet the former areviable D. simulans w- P[/ac-w+]insertion - and the latter are inviable. Therefore, this result pro- vides strong evidence thatinviability fktors arepartially or fully recessive. R- ‘:ph The Fact thatthe unbalanced female experiment D. simulans D. mauritiana gives different results for fertility and viability has led to the suggestion that HXI.I)ANE’S rule is a composite phenomenon with different Factors affecting the sex- FI hybrid / specificexpression of hybridsterility and inviability (ORR 199311;M‘u and DA\‘IS1993). HALDANE’Srule with respect to inviability can be explained by the recessivity of incompatibility Factors, whereas the sex-specific fertil- ity difference may have a different genetic basis. Wu : repeatedbackcrossing and DA\TS(1993) have suggested that male fertility fac- i for 15 generations tors in Drosophila may evolve more rapidly than female fertility Factors and thereby contribute to the large num- ber ofhybridizations in whichmales are sterile and t. -1 females are fertile. 2x = 4.7- cM after 15 generations X More empirical data are needed to settle theseissues about the geneticbasis of HALDANE’S rule and the large Distriiution of intact segment length,x X effect. Here we report a Drosophila experiment that 0.15 addresses the following questions: (1) Do male sterility * mean =4.7 factors accumulate at different rates on the X us. au- tosomes? (2) Do male and female sterility factors accu- mulate at differentrates? (3)Do fertility and inviability Factors accumulate at different rates? The species used in this experiment are I). simulons and D. mmwitioncr, which (together with their sibling species 11. srrlwllicr) are theclosest known relatives of 11. mrlanogastw (/kSHRC1RNI:.R 1989). 11. m,trztri/ionrr is en- demic to thc island of Mauritius, while simdons has an essentially cosmopolitan distribution, but has not been found on Mauritius (LWHAISEPI ol. 1988). These two species are very closely related. They diverged.” fromone .. -“-,“m-mmr.m-mvll-m another “0.6-0.9 mya (HEYand KI.IMAN 1993), they * -““NNNNN have a Nei’s geneticdistance of 0.3 (CARIOU1987), m in cM and they have homosequential polytene chromosomes FIGURE1.-The procedure by which a P-element marked (LEMEL~NIERand A~HRURNER1976). The cross of sim- segment of the mnurifinnn genome was introgressed into a zrlons females to mnuritinna males goes easily, while the simulans hackground by repeated hackcrossing. The I’ insert reciprocal cross is difficult. In both crosses, the FI hy- contains a mini-zohilr gene (V),which provides an eye color brid females are ftllly fertile and can be backcrossed to marker used for selection of heterozygous females as parents for each backcross. After 15 generations, the length of the either species, while the males are completely sterile, introgressed segment on each side of the marker is expected thereby providing a classic example of HALDANE’Srule. to have an exponential distribution (as shown) with a mean In addition, previous work involving a standard back- of 4.7 cM (see APPENDIX). cross analysis has demonstrated the large Xeffecttypical the simulnns males for 15 generations (Figure I). At this point, of many other species pairs (C<)\’NE1984). Therefore, a segment of the mnuritiann genome remains associated with this species pair is well suited for investigation of these the marker, while the rest of the genome is almost entirely general phenomena. simulnns. For autosomal segments,a single heterozygous male offspring was chosen to generate homozygous males and fe- males for viability and fertility testing,which permit5 detection of recessive factors affecting hoth males and females (Figure MATERIALS AND METHODS 2). For X-linked segments, a single hemizygous male was cho- sen to generate homozygous females, thereby testinghis fertil- Experimental design: Here we use anintrogression ap ity. If he was fertile, homozygous females were also tested for proach to study hyhritl sterility and inviability factors on hoth viahilit! and fertility (Figure 3).Numerous marked segment5 the X and autosomes. Briefly, males from a mnwitinnn stock fromboth the X and autosomes weretested in this way, containing a semidominant marker were crossed to simulnns allowing comparisons of fertility for hemizygous X segments females and hyhritl females were repeatedly backcrossed to and homozygous autosomal segmentsof similar average size. 822 J. R. True, B. S. Weir and C. C. Laurie

FIGURE2.-The procedure for making ho- mozygotes of an autosomal introgression and for testing their viability and fertility. The out- come of viability and fertility tests are shown using the same symbols as in Figure 4. The viaFle triangle on the chromosomerepresents the first eeneration inviable p[lac-ru'] insert. The labels A1 -A8 are defined in MATERIAIS AND METHODS.

later generation(s) fertile

This experiment was originally designed for generating Each of the Pinserts in these stocks was localized by in situ fixed, homozygous introgression lines for the study of mor- hybridization tothe polytene chromosomes. Those inserts phological differences between the species (and not specifi- mapping to the same cytological location were pooled and a cally for the study of hybrid incompatibilities). Thus, the via- single representative of each pool was used for constructing bility and fertilitytests are a byproduct of the process of a genetic map by estimating the recombination fraction be- attempting to establish fixed lines. The criterion used to assess tween pairs of inserts at adjacent positions. The total map fertility was an ability to produce offspring in a cross. Because length between the most extreme P markers is 468 cM and a many different crosses werehandled in a short period of time, total estimate that also includes the unmarked terminal re- it was not possible to assess sperm motility in males or to gions of each chromosome is 505 cM. Further details about confirm that sterile females had been inseminated, as is fre- the Pelementline constructionand the genetic mapare given quently done in hybrid incompatibility studies. Therefore, by TRUEet nl. (1996). sterility in this study includes both failure to produce func- Distribution of P-element markers: The 103 independent, tional gametes as well as failure tomate and/or transfer homozygous viable and fertile insertions used in this study sperm. It is unlikely that male sterility is caused by mating are distributed across all four of the chromosomes at a total discrimination, because fertility tests involved mating hybrid of 87 distinct cytological positions, as shown in Figure 4. To males to simulnns females, which readily mate with males of determine whether these insertions are randomly distributed either species (COYNE1989). However, some cases of female among the three major chromosomes, a chi-square test was sterility may be due to a failure to mate, because maun'liana performed using expected numbers calculated on the basis females discriminate strongly against simulanc males (RORERT- of the polytene band number length of each chromosome. SON 1988) and some introgressions may contain the genes When all of the independent insertions are considered, the that control this behavior. test is significant (0.005 < P < 0.01). and whenonly the Drosophila stocks: The experiment was initiated with a 87 distinct cytological positions are considered, the test is 7uhitF mutant stock of each species, both provided by J. A. borderline significant (0.05 < P< 0.10). There is a deficiency COYNE. The initially outbred simulans 7u- stock (13w) was of inserts on the second chromosome relative to the others. taken through 20 generations of full sib mating to produce an Another test was performed to determine whether the in- inbred 70- stock, which was then maintained by mass transfer. sert positions are randomly distributed within each chromo- Construction of P-element marker stocks: To provide a set some arm. Each cytological location was assigned a numerical of semidominant markers distributed throughout the ge- position on the chromosome based on its polytene band num- nome, the 71)- maun'tinnn stock was transformed with a mini- ber, and the average of all pairwise differences between the white Pelement construct, p[lnc-71~+](BIER rt nl. 1989). by positions within an arm was calculated. This measure is the embryo injection. Single insert transformants were selected Gini dispersion (STUARTand ORD1987). which has an ex- for further use by a Southern hybridization analysis and then pected value of L/3 for a uniform distribution on interval established as homozygous stocks by intercrossing the prog- (0,L). To test the significance of the difference between oh eny of a single heterozygous fly. Only homozygous viable and served and expected for each arm, 1000 sets of n points were fertile inserts were used for the experiment reported here. In placed randomly on a lineof length L, where n is the observed the process of establishing these stocks, a large chromosomal number of insert positions and I, is the polytene band length. segment linked to the I' insert marker is made essentially The Gini dispersion was calculated for each set and the sig- isogenic, thus eliminating any preexisting lethals and steriles nificance level was calculated as the proportion of the 1000 that may have been segregating within the host stock. sets that had a difference between observed and expected Hybrid Incompatibility in Drosophila 823

backcross 15 1-1 - x -a -J.

viable B x -3 1

fertility test

FI(;I'KI.3.-Thc proccdurc lor making homozygotrs antl hcmizygotcs ofan X-linker1 introgression and for testing their viability antl f+rtilitv. Thc oIItcoInc of viability and ftv-tilitv tests are shown using the same symbols as in Figure 4. The triangle on the chromosomr rcymwnts thc P[/f7f-711t] insert. Thc- labels XI -X4 are defined in M..\TERIAI"-i ,\\ND MF.TII0DS.

within the I'[/rrc-~o+]construct provides an eve color marker that varies in expression among insert5 due to position effects. In most cases, it provides a semidominant phenotype such that flies with no copies of the insert have white eyes, heterozy- gous flies have light orange eves and homozygous flies have dark orange or red eves (but not fullywild type). Inmost lines, males show a darker eye color than females. There was never any clifficrdty in distinguishing flies with no inserts from those having one or two, but test crosses were often needed to verify the distinction between heterozygotes and homozy- gotes forthe insert. In the follo~~ngdescription, orangerefers to putative heterozygotes and red refers to putative homozy- gotes for the insert. Viabilityand fertility testing of autosomal introgres- sions: \'iabilitv and fertility tests were made during the pro- cess of attempting to make the /' insert homozygous within each slhlinc (Figure 2). Three or four orange male progeny from the 15th backcross were individually mated to 71)- sim- zrhsvirgin females (cross Hl). The progeny from just one of the fertile males were used to set up the next cross of orange femalrs by orange males (cross H2). If red progeny of both sexes were produced, a red by red mating was set up (cross HS). If the red by red cross was fertile, the subline was monitored for several generations to verify fixation of the P insert. For the majority of sublines, there were problems at one or more steps of this procedure due to inviability, sterility or difficulty in distinguishing flies heterozygous or homozygous 824 1. K. True, R. S. \I’c.ir and (:. C. 1,auric ch ro m o som e chromosomeX chromosome 4 8 8 N BF! !& ;,.,;,Bo 0,... . I 1214 1319 18 175 16 110 111 112 114113 11s117 116 11 +?I A no. 0 DADA A .A. Y. ~mmn A P A .rn m1.m . 19 101 . 20 A. A = 1.. ...A. A .. 11 .A .A. .A chromosome 2

0 0 10 0 00 0 0 0 0 00 8 00000 00 8 21 22 2324 25 262728 29 30 131132133 134 135 136 137 138 139boq 142143 144 145146147 148 149 150 151 152 153154 155 156157 158 159160 P 0. P .00 m mn 0 4: : . 0... . 0.. . . . A. . g rg 8‘ ’ i: . chromosome 3 i B 00 0 X 80 X1 ! +8 0 v? !ji, !I8 0 .is 0 , I B I::0 , b 08 fi@ 61 162 163 164 165166167168 169 170 171172173 174175 176177178 1791 62183 1845931 921911 901891 881 8718618518194 195196 197 198 199 11 no P uno nun ran a 0. 00 an 0 i ii ‘0.Iljo YO EO PO . . 0 .....a .O .. . ..O 0 P . O 5 . 0 2 io ig i:: .. y 6 io oo

Xchromosome Autosomes o (XI) single male fertile (AI) both sexes fertile in fin1 cross at generation 16 (X2) single male sterile. mass male fertile 0 (A2) weak viability. but tint cross fertile at >generation 16 (X3) single and mass male sterile 0 (A3) first cross slerile.but subsequent crosses fertile A (X4) single male sterile, mass male untested (A4) male sterile 0 (A5) female sterile A (A6) both sexes sterile 0 (A7) both sexes inviable 0 (AB) very low viability when both parents homozygous

FI(X.RI<4.”The clistrihutioli of introgression srddincs showing dif’fi-rrnt types of incomp;~til,ilitics. hch symhol rcpresents a singlc sublincpositionrtl at the cytological location of its Pclement insert. The eight ;wtosomal and four X chromosome categories arc tlcscrilx-tl Inore f’i~llyin JI..VIIlinewas classified as 143. werr proven lbrtilc in 21 tc’stcross to 70 .si~~rrt/~ttr.s~~lal~~s.(In some AS: The rrtl by ~-cdcrosscs (involving an average of 6.2 fe- C~SCSa subline was lost l)c*forc.all trsts could IIC completctl.) m;lles and 10.1 males) ncvcr )icldetl progeny, hut both red ,.\S: All ~-cdby red crossw attempted (involvitlg an average Inales antl red f+malcs wrc fertile in trst crossrs to siurrtlnns 7K. Hybrid Incompatibility in Drosophila 825

The backcross control introgressions were taken through subline. For X-linked run, a mass homogenate of 10 hemizy- the same procedure for making homozygous lines as the au- gous males from each subline was analyzed. For each of the tosomal P introgressions (Figure 2), except that parents for autosomalintrogressions, there were 15 backcross genera- each cross were randomly selected. These lines provided a tions before the molecular analysis. For run, many of the lines total of 64 single male fertility tests of which 91% were fertile. analyzed were male sterile and backcrossing was continued This fertility level is very similar to that for the heterozygous for additional generations before the molecular analysis (an autosomal inserts,for which a total of 881 single male matings average total of 21 generations). were 88% fertile (cross H1, Figure 2). These figures provide a baseline for judging the reliability of the fertility tests of homozygous and hemizygous males. If the probability of steril- RESULTS ity due to genetic background or heterozygosity for an intro- gressed segment is 0.10 for a single male, then the probability The length of introgressed segments: An important that three tested males are all sterile is 0.001. question in the interpretation of this introgression ex- Viability and fertility testing of X chromosome introgres- periment is how much of the mauritiana genome re- sions: The procedure for making X chromosome sublines homozygous is summarized in Figure 3. Three or fourhemizy- mains associated with the selected P-element marker gous red male progenyfrom the15th backcross were individu- after 15 generations of repeated backcrossing to sim- ally mated to w- simulunsvirgin females (cross Hl). Theprog- ulans. This amount clearly depends on the level of re- eny from just oneof the fertile males were used to set up the combination in the hybrid flies during the repeated next cross of orange females by w- simuluns males (cross H2). backcrossing procedure.The results of a molecular Then orange female and hemizygous red male progeny were crossed (H3)to obtainred females and hemizygous red marker analysis in the APPENDIX indicate that the effec- males. If the red by red cross (H4) was fertile, the subline tive level of recombination is only about one-fourth of was monitored forseveral generations to verify fixation of the that expected on the basis of the mauritiana genetic P insert. map. Previous work shows that the mauritiana genetic Many of the sublines could not be fixed because of male map is about 1.4 times larger than the simulans map fertility problems. Each X chromosome subline was classified into oneof four categories, according to thecriteria described and, for each of four homologous intervals analyzed, below. F, hybrids have a recombination level within the range XI: At least one of the single male matings (Hl) was fertile. of the two parents(TRUE et al. 1996).Thus, such a Among 26 sublines classified as X1, 18 produced fertile red large reduction in the apparent level of recombination females that were used to establish fixed sublines and seven during introgression was not expected. were lost before homozygous female viability and fertility could be tested. One subline failed to produce red homozy- To determine whether the actual level of crossing gous females and is potentially a female-specific inviability over differs between the introgression lines and the factor. (In this case 18 females of the darkest eye color avail- original Pinsert lines in mauritiana, recombination frac- able were proven heterozygous in a cross). tions for a sample of 12 intervals were estimated as X2: Sublines for which all of the single male matings (Hl) previously described (TRUEet al. 1996). Table 1 shows were sterile, were maintained forseveral generations and then thatthe recombination estimates in pure mauritiana fertility tested again by mass mating an average of 9.3 hemizy- gous red males to w- simuluns females (H5). Sublines were flies are consistently higher than in the introgression classified as X2 if the mass mating was fertile. flies and most of the pairs show a highly significant X3: Sublines were classified as X3 if the mass mating test difference. The ratio of the recombination fraction in (H5) was sterile. introgression flies to that in mauritiana flies varies from X4: Several sublines had low viability and were lost before 0.16 to with a mean of 0.45. Although the geno- the mass mating test could be done; these are classified as X4. 0.83 Molecular marker analysis of introgression lines: Several types of the introgression flies used for estimating re- molecular markers that distinguish the mauritiana and sim- combination fraction (trans heterozygous for partially uluns stocks were analyzed to obtain information about the overlapping introgressed segments) are not quite the length distribution of introgressed segments. These markers same as those during the introgression process, these are restriction site differences of polymerase chain reaction- results provide evidence that crossing over was indeed amplified fragments, which were developed and analyzed as described by LIU et ul. (1996). Briefly, primer pairs were de- lower overall and that the degree of difference varies signed on thebasis ofD. melunogustersequence data, fragments among chromosomal locations. were amplified from simuluns and muuritiunu, and restriction The fact thatthe ratio of recombination level in- site differences were located by sequencing. For genotypic ferred from the molecular markers is -0.26, whereas analysis, fragments amplified from genomic DNA were mixed that estimated by direct measurement of crossing over with a control fragment (toverify restriction enzyme activity), digested and then analyzed on either agarose or acrylamide is 0.45, suggests that there may have also been a bias in gels. Among 18 markers tested, only five were found to be the recovery and persistence of recombinant chromo- fixed for different alleles in the muuritiunu and simuluns stocks somes in the hybrid flies during introgression. A priori, used in this experiment (on the basis of five different mass one might expectselection to favorsmaller introgressed fly homogenates per stock). These are jun (99D), h (66D), segments because of hybrid incompatibilities. However, Antp (84B), sli (52D) and mn (19E). A number of fixed au- tosomal introgression sublines in the vicinity of a molecular the effect of selection actually appears to favor the re- marker were analyzed to determine whether they carry the tention of longer introgressed segments, perhaps be- mauritiunu or simuluns allele. For this purpose, genomic DNA cause of inbreeding depression in the recurrent sim- was prepared from a mass homogenate of 20 flies from each ulans parental stock. There is some evidence that this 826 J. R. True, B. S. Weir and C. C. Laurie

TABLE 1 Recombination fraction comparison between pure mauritiam and introgression lines

RF RF Interval mauritiana" itnrogression" Ratio x' (1 d.f.) P value 3A-4B 0.198 0.037 0.20 125.87 P < 0.005 26B29CD 0.092 0.057 0.62 12.37 P < 0.005 34DE-35F 0.084 0.070 0.83 2.28 NS 42B-44D 0.113 0.028 0.25 53.99 P < 0.005 47F-52DE 0.224 0.096 0.43 91.56 P < 0.005 52DE-57A 0.182 0.136 0.74 10.19 I' < 0.005 61CD-62E 0.093 0.025 0.27 65.97 P < 0.005 65A-66B 0.104 0.017 0.16 88.76 P < 0.005 0.087 0.057 66B67DE 0.087 0.66 10.90 I' < 0.005 84DE-91A 120.23 0.151 0.20 0.030 P < 0.005 97A-98DE 0.131 0.043 0.33 64.45 P < 0.005 98DE-99D 0.075 0.052 0.69 6.86 0.005 < P < 0.01 NS, not significant.

" Average sample size per recombination fraction (RF) estimate = 3274 flies. was in fact the case. During the first 10 generations of Virtually no incompatibilities were detected for het- introgression, aninbred simulans stock was used (to erozygous segments. Although a number of sublines facilitate subsequent molecular marker analysis), but were lost during the process of introgression (44 out because ofdifficulty in maintaining the lines, it was of a total of 399 = 11 %), at least one subline from each replaced at generation 11 with the outbred stock from of the original Pinserts survived the 15 generations of which it was derived. introgression, indicating there are no dominantfactors In the APPENDIX, we conclude that the genome of an causing female inviability or sterility, as expected, be- introgression line after 15 generations of backcrossing cause F, female hybrids are fully viable and fertile. All is essentially pure simuluns except for anintact segment autosomal sublines gave viable heterozygous males and of mauritiana chromosome surrounding the selected P all X-linked sublines gave viable hemizygous malesafter insert. The segment lengthis expected to have an expo- 1.5 generations. In addition, all but two of 277 autoso- nential distribution with a mean value equal to -7% mal sublines tested gave at least one fertile heterozygous of the genetic length of the hybrid genome (which is male. The overall level of heterozygous male fertility -35% of a major chromosome arm). It is important to was quite high (88% of 881) and similar to that of notethat this distribution is asymmetrical such that the control introgression lines, which had no selected small size classes havehigh probabilities of occurrence marker (91% of 64). Therefore, essentially all of the relative to larger size classes (see Figure 1). It is also incompatibilities detected here are recessive in terms important to note that the effective level of recombina- of causing complete sterility or inviability (although tion (and therefore the length distribution) probably they may also have some deleterious effects when het- varies among different chromosomal regions. There- erozygous and could even be partially dominant on a fore, the rangeof length variation is likely to be consid- continuous scale of fertility measurement). erable. The chromosomal distribution of the incompatibility Types of incompatibility: After 15 generations of in- categories is shownin Figure 4. In this figure, each trogression, an attempt was made for each subline to subline is represented by a symbol denoting its category, established a fixed stock homozygous for the P insert which is placed at the cytological position of its P ele- (Figures 2 and 3). This should have been possible in ment insert. Symbols shown above the X chromosome the absence of interspecific incompatibilities (and represent lines that were fertile in the single hemizy- newly arising lethal or sterile mutations), because each gous male tests. Many of those sublines were also homo- of the P inserts is homozygous viable and fertile in a zygous female fertile, although several were lost before mauritiana genetic background. However, a majority of female testing. Symbolsshown above the autosomes sublines manifested some type of incompatibility that were homozygous viable and fertile in both males and prevented the establishment of a fixed stock. In addi- females on the first attempt. Other sublines below the tion, many of the fixed lines are weak and difficult to autosomes (category A3) were eventually male and fe- maintain. Depending on the type of problem encoun- male fertile also, but only after one or more opportuni- tered during the fixation process, each of the sublines ties for further recombination with a simulans chromo- was classified into one of eight autosomal or four X- some. linked categories, as described in MATERIALS AND Comparison of Xand autosomal effects on male ste- METHODS. rility: A controversial issue in the study of hybrid in- Hybrid Incompatibility in Drosophila 827 compatibility genetics is whether the X chromosome TABLE 2 typically contains a higher density of male sterility fac- Comparison of sterility levels in X us. tors than the autosomes. One of the requirements for autosomal introgressions answering this question is to define an appropriatemea- sure of that density. It is probably not possible to actu- Total Total ally count the numberof discrete factors because of the Chromosome Fraction number number arm male sterile" sublines fact that, on the Xchromosome at least, there appears P positions to be a large number of factors that interact in compli- X 0.7593 22 cated ways to produce sterility (WU and PALOPOLI 2L 0.66 11 39 1994). However, the introgression design provides0.41 an 2R 11 35 alternative approach that is based on comparing the 3L 760.45 21 3R 0.57 20 73 phenotypic effects of introgressed segments of equiva- 4 0 2 4 lent size from the twotypes of chromosome. It also provides a meansof comparing Xchromosomalhemizy- For the X, F = (X2 + X3 + X4)/(Xl + X2 + X3 + X4). For the autosomes, F = A2 gotes with autosomal homozygotes, which is necessary (A3 + A4 + A6)/(Al + + A3 + A4 + A5 + A6 + AS). The autosomal calculation assumes because of the fact that many sterility factors could be that the failureof the first fertility test in A3 sublines was due recessive. to male sterility. One measure of the relative density of male sterility "The fraction of sublines (F) at each P position that are factors on a chromosome is the probability that a ran- male sterile was calculated from the number belonging to each category,as shown in Figure and definedin MATERIALS domly placed segment from maun'tiana confers sterility 4 AND METHODS. in an otherwise simulans genetic background.The intro- gression lines can provide such a measure as long as the selected P inserts are randomly located along the by the molecular marker data. (2) During the process length of the chromosome. As described in MATERIALS of making an autosomal introgression homozygous, the AND METHODS, there appears to be a deficiency of P cross between heterozygous males and females (H2 in inserts on the second chromosome relative to the oth- Figure 2) provides an opportunity for recombination ers, but within each chromosome arm, the insert loca- in the female that can result in heterogeneity in the tions do notdiffer significantlyfrom a randomexpecta- lengths of the segment within a subline. The fertility of tion. Thus, the probability of sterility resulting from a Xchromosome sublines was assessed before the compa- randomly placed maun'tiana segment can be estimated rable step (cross H3 in Figure 3). This difference will as the fraction of sublines with their Pinsert ata particu- tend to make the completely homozygous portion of lar location that aresterile. For example, if two of three the introgression somewhat smaller than it was after independent sublines for the same P insert are sterile, just 15 generations of backcrossing. However, the differ- the fraction for that P position is '/=+The density of ence in expected lengthbetween 15 and 16 generations sterility factors on a whole chromosome can be charac- is only 6%. (3) TheAPPENDIX analysis provides evidence terized by the average of this fraction over all of the for heterogeneity in the distributionof segment lengths different P-insert locations. This measure of density re- among different chromosomal regions. If such hetero- flects both the number of factors and the relative mag- geneity exists, the results of a single X chromosome nitude of their effects. One segment may produce steril- marker couldprovide a misleading picture of that chro- ity because it has a single factor with large effect, while mosome. (4) Although the maximum likelihood esti- another segment may produce sterility because it has mate of the recombination parameter (Table Al) for multiple factors with cumulative effects. a marker in the proximal Xis similar to that for two of A comparison between chromosomes of the average three autosomal markers, direct measures of recombi- fraction of sterile sublines is valid onlyif the distribution nation in introgression lines (Table 1) show that a sin- of introgressed segment lengths doesnot differ between gle interval in the distal X has a greater reduction in chromosomes. The analysis of segment lengths in the recombination relative to thepure maun'tiana value APPENDIX involves a comparison of one Xchromosomal than most of the autosomal intervals analyzed. Thus, region with four autosomal regions. There is no com- the assumption of homogeneity in segment length dis- pelling evidence that the X differs consistently from tributions between the Xandautosomes is questionable the autosomes, but there are four complications that and conclusions from the following analysis of X us. should be considered. (1) There is a potential bias in autosomal effects should be tempered accordingly. the molecular marker data in that thesublines analyzed The average fraction of sterile sublines at a particular for theXchromosome markerwere mostlysterile, while location is given for each chromosome arm in Table 2. the autosomal sublines were nearly all fertile. If sterile The average value forthe X (0.75) is considerably introgressions are typically longer than fertile ones(NA- higher than the overall autosomal average (0.50) (as \TIM 1992), then theaverage of the autosomal segment well as the average for each autosomal arm). To deter- lengths may actually be somewhat larger thanestimated mine whether this X-autosomal difference is statistically 828 J. R. True, B. andS. Weir C. C. Laurie

TABLE 3 the X and found that females heterozygous for male Comparison of male and female fertility in autosomal sterile segments are significantlyless fertile than fe- introgression sublines males heterozygous for male fertile segments (JOHN- SON and WU 1993). This result suggests a possible rela- Male Male Male tionship between male and female incompatibility fertile sterile untestedTotal factors. However, an alternative interpretation is that Female fertile 112 53 - 165 male sterile introgressions may tend to be longer than Female sterile 6 4 2 12 male fertile ones (NAVEIRA1992) and therefore also Female untested - 8 - 8 more likely to contain female sterility factors. Total 65 118 2 185 Inviabilityfactors: Twotypes of inviability factors The fertile and sterile classifications are definedwith refer- were detected among the autosomal lines. In one case ence to the categories described in MATERIAIS AND METHODS. (category A7), red homozygotes were never obtained. Male fertile categories are AI, A2, A5, and A8. Male sterile There were nine such sublines at five different chromo- categories are A4 and A6. Female fertile categories are Al, somal locations out of a total of 185 sublines (5%), A2, A4, and A8. Female sterile categories are A5 and A6. which is about the same frequency as female steriles. The other type of inviability factor detected was classi- significant, a permutation test was performed. There fied as category AS, in which homozygotes are lethal or are 22 Xchromosome locations and 65 autosomal loca- have very low viability, but only when both parents are tions in the dataset. The sterile fraction values for these homozygotes. There were 17 A8 lines at 14 different 87 locations were pooled, a sample of 65 values were chromosomal locations (9% of autosomal sublines). chosen at random from the pool, and the average value The fraction of autosomal sublines showing one or the for this set was subtracted from the average for the other type of inviability is 14%, which is less than half remaining 22 values. The significance level of the test the fraction of viable sublines showing male sterility. is the fraction of these random sampling events that The A8 category phenotype is consistent with a mater- give an average difference greater than or equal to the nal effect lethal showing zygotic rescue. The fact that observed difference of 0.25. A set of 1000 permutations homozygous inviability is manifested only when the gave a significance level of 0.003. Given the assumption mother is also homozygous suggests that a necessary of segment length homogeneity, this result indicates gene product can be provided either maternally or zy- that a mauritiana segment randomly placed on the X gotically. This phenotype is similar to that for several chromosome is more likely to confer sterility than a previously described D. melanogastermutants,such as cin randomly placed segment of equivalent size on an au- (BAKER1973). tosome. There are two unexpected casesof clustering that Comparison of male and female sterility factors: A involve inviability factors. All four of the sublines for comparison of male and female sterility factors on the the insertat 92BC show typeA7 inviabilitywhereas none autosomes is straightforward because each autosomal of the six sublines for the nearby insert at 92E show introgression that was viable in both sexes (the major- inviability. Similarly, three of the four sublines for the ity) could be tested for both male and female sterility insert at 24A show type A8 inviability whereas none of (although a few sublines were lost before both tests the three sublines for nearby 24CD showinviability. could be completed). Table 3 shows that among 185 One plausible scenario to account for these observd- homozygous viable autosomal sublines, only 7% are fe- tions is that there was polymorphism of incompatibility male sterile whereas 36% are male sterile, which is a factors within the mauritiana host strain at the time of highly significant difference (x:= 44.2). An average of P-element transformation and different alleles became 5.4% of sublines per insert location are female sterile associated with different P insertions in the same re- compared with 50% for male sterility. Thus, it is clear gion. Unfortunately, relatively little is known about the that the autosomal density of effects on male sterility level of intraspecific polymorphism in genes that confer is muchgreater than that on female sterility. For X hybrid incompatibility (but see WU and PALOPOLI introgressions, the high level of male sterility prevented 1994). the majority of sublines from being tested for homozy- Spontaneous mutationduring the experiment: gous female fertility. However, all 18 X sublines tested There is a possibility that some of the incompatibilities were female fertile, so it appears that female sterility detected in the introgression lines are actually due to factors are also scarce on the Xchromosome. recessive deleterious mutations that either preexisted Among the 10 female sterile autosomal sublines that in theoriginal mauritiana stock or arose by spontaneous were tested for male fertility, four were alsomale sterile, mutationduring the course of theexperiment. Pre- which is about the number expectedby chance if male viously existing mutations capable of causing sterility or and female sterility are independent (3.2). A previous inviability on aconspecific background arevery unlikely study examined the quantitative level of female fertility because a relatively large region surrounding each P in three pairs of mauritiana/simulans introgressions on insert was made essentially autozygous during stock con- Hybrid Incompatibility in Drosophila 829 struction (see MATERIALSAND METHODS). However, new cross studies is justified. Therefore, it is clear that back- mutations may have accumulated during the15 genera- cross data cannot be used to detect a differencein the tions of introgression during which the mauritiana seg- density of sterility effects betweenthe X and autosomes ment was constantly heterozygous and, therefore, shel- because of the confounding effects of dominance. tered from selection. The introgression design used here removes the con- Previous estimates of the spontaneous rate of reces- founding effects of dominance and provides the oppor- sive lethal mutation in D. melanogaster average -0.005 tunity to detect differences in density between chromo- lethals per second chromosome per generation (SIM- somes. However, the absolute magnitude ofthe MONS and CROW1977). The rate for a segmentwith an difference cannot be estimated for two reasons. First, average length -18% of the second chromosome (i.e., the probability that an introgressed segment confers the average introgressed segment) is (0.18) (0.005) = sterility depends on both the number of factors and 0.0009 and theprobability that no lethal mutations have the magnitudeof their effects, asnoted earlier. Second, occurred in this segment during 15 generations is 0.99. the ability to detect chromosomal differences in density Therefore, - 1% of introgressions are expected to have depends on the average size of introgressions. Large a new lethal mutation compared with 5% for type A7 segments frequently may include more factors than are inviability inthis experiment. Type A8 inviability would necessary to confer sterility and small segments seldom not be scored as a lethal in the typical mutation rate may include enough factors to confer sterility (NAVEIRA experiment. Thus, the true level of hybrid inviability 1992; Wu and PALOPOLI1994). Therefore, thefact that may be less than the 5% of sublines observed and it is X introgressions are 50% more likely to confer sterility even possible that all of the A7 inviability casesare due than autosomal introgressions does not necessarily im- to new mutations. ply that the density of factors is 50% greater. In mutagenesis experiments, the rates of occurrence Now we consider whether thelarge X effect observed of male and female steriles are each -10-20% of the in the backcross analysis of D.simulans and mauritiana lethal rate (LINDSLEYand LIFSCH~~Z1972; COOLEYet al. (COYNE1984) is due to dominance effects and/or a 1988; CASTRILLONet al. 1993). So an estimate of the difference in the density of sterilityeffects. In these spontaneous rate for steriles is roughly (0.15) (0.005) = species, the X chromosome has a euchromatic length 7.5 X lop4 per second chromosome. Using this rate, approximately equal to one major autosomal arm. The only -0.2% of introgression lines are expected to have null hypothesis is that all incompatible gene pairs have either a new male or female sterile mutation. There- the same effect whether hemizygous, homozygous or fore, essentially none of the hybrid sterility detected heterozygous, and that the effects are additive among can be attributed to new mutations. gene pairs. Under this hypothesis, if the density ofsteril- ity factors on the Xandautosomes is equal, abackcross DISCUSSION male with a mauritiana X and all simulans autosomes should have the same sterility level as one with a single Comparison of X and autosomal effects on male ste- heterozygous mauritiana autosomal arm and the rest rility: This introgression experiment shows that re- simulans. However, the former genotype is completely placement of a segment of the simulans genome with sterile while a significant fraction of males of the latter the homologous segment from mauritiana is more likely genotype are fertile (an average of 16.2%for genotypes to cause male sterility when the segment is X chromo- 3-6 of COYNE1984). Therefore, the X has a greater somal than when it is autosomal, given two assumptions. effect either because it has more sterility factors or the These assumptions are that the P inserts are randomly effects of those factors are greater (perhaps because distributed within chromosome,a which wellis they are hemizygous while the autosomal factors are founded, and that thesize distribution of introgressions heterozygous). Even heterozygosity for three autosomal does not differ between the X and autosomes, which arms permits a fertility level that is significantly greater is questionable. Therefore, the results presented here than the single Xsubstitution (average of 2.2% for ge- provide only a suggestion that the X and autosomes notypes 9-12 of COYNE1984). When all four autosomal differ in density of effects on male sterility. Additional arms derive from mauritiana, three of 222 males were work is needed to verify this suggestion. fertile, but this is not significantly different from the Several previous backcross studies havealso sug- single X substitution (0 of 187 fertile) (genotypes 14 gested a largerdensity of Xchromosome factors (COYNE and 2). Therefore, the Xwould have to contain about and Om1989a), butthose studies involved comparison four times as many factors as the autosomes to account of hemizygous X with heterozygous autosomal seg- for these observations under the null hypothesis. ments, which provides a detection bias in favor of the Are the introgression results presented here consis- X chromosomewhen autosomal sterility factors are not tent with a fourfold higher density of factors on the X completely dominant. Here we find autosomal factors than autosomes? As discussed above, the results indicate that are fertile when heterozygous but sterile when ho- that Xintrogressions are -50% more likely to be sterile mozygous, showing that concern aboutthis biasin back- than autosomal introgressions, but this figure could 830 J. R. True, B. S. Weir and C. C. Laurie represent asignificant underestimate of the actual den- not apply to all Drosophila species. Similar studies of sity difference. Therefore, although the data suggest a additional species must be done before reaching any fourfold difference is unlikely, the hypothesis cannot general conclusion about X us. autosomal differences. be rejected. Theoretical studies have explored conditions under An alternative explanation of the large X effect is which X linked lociwould evolve more rapidly than that gene interactions involving a hemizygous X factor autosomal loci (CHARLESWORTHet al. 1987; COYNEand generally have greaterdeleterious effects than those ORR 1989a).The basic finding is that the rate of substi- involving a heterozygous autosomal factor (i.e., recessi- tution on the Xwill be greater than the autosomal rate vityas in the X-autosome imbalance and dominance if new advantageous mutations arerecessive or partially theories of HALDANE'Srule). While the attached-X ex- recessive on the average, as long asthey affect male periments of ORR (1993b) provide strong evidence of fitness. However, the authors note that there are two recessivity for inviability factors, the evidence for fertility difficulties with this theory. One is that there is no em- factors (COYNE 1985) is inconclusive, as noted earlier. pirical evidence that the advantageous effects of alleles Although it would not be surprising if inviability and that later cause interspecific incompatibilities are fre- sterility factors have similar dominanceproperties, quently recessive. Another is that the large Xeffect (as there arereasons to suspect they may not. For example, detected in backcrosses) is consistently observed for X-autosome translocations in D. melanogaster are usually hybrid sterility, but not for other types of characters dominant male steriles whereas this is not the case for such as premating isolation or morphological traits either viability or female fertility (LIFSCHWZand LIND- (CHARLESWORTHPt al. 1987). It is not clear why there SLEY 1972). Therefore, directevidence concerning the should be a difference in the dominance relations for dominance of fertility factors is needed. different types of traits, but CHARL.ESWORTHel al. (1987, Unfortunately, the most we can conclude from direct 1993) have suggested that genes that interact negatively evidence is that male sterility factors are neither com- to produce hybrid incompatibility may also tend to pletely dominant nor completely recessive. The intro- show nonadditive interactions between alleles within a gression results clearly showthat autosomal sterility fac- locus. tors arenot completely dominant, since nearly all Comparison of male and femalesterility factors: heterozygotes are fertile while many homozygotes are There is a clear difference in the effects of introgressed sterile. The fact that autosomal male sterility factors autosomal segments on male and female fertility. An are not completely recessive can be inferred from the average of only 5.4% of sublines per insert location are backcross experiments of COYNE (1984), which show female sterile compared with 50%for male sterility. that segregating autosomal genotypes have significant These results are consistent with those of H. HOL effect on male fertility in the backcrosses of F1 females LOCHER and C.-I. Wu (personal communication) who to both maum'tiana and simulans. The critical question constructed second chromosome introgressions involv- for explaining both the Xeffect and HALDANE'Srule is ing the same two species. Although their approach was whether the degree of dominance is less than one-half. quite different, they also found a greater frequency of An answer to this question probably will require a quan- male than female sterility in lines homozygous for mau- titative measure of the fertility of individuals, rather m'tiana segments within a simulans background. than a simple qualitative assessment of fertility or steril- Although the comparison between frequencies of ity. Meanwhile, the cause of the large X effect in Dro- male and female sterile autosomal introgressions in- sophila backcross studies remains an open question. volves much less uncertainty than the X-autosome com- The X us. autosomal difference observed in our study parisons, there is a possibility that the observed differ- may reflect a differential rate of substitution per locus ence is somewhat biased by featurea of the within each species during their geographic isolation. experimental design. Because, within each subline, sev- This inference depends on two assumptions. (1) The eral hybrid females were backcrossed to szmulans each density of loci capable of affecting hybrid fertility is generation, there is a possibility of selection against similar on the X and autosomes. This assumption can introgressed segments containing female sterility fac- be supported if hybrid sterility factors represent genes tors that are notcompletely recessive. In addition, 11% that normally play a role in spermatogenesis, because of sublines were lost during the 15 generations ofintro- mutagenesis studies in D. melanogaster have previously gression. Selection against heterozygous mauritiann fac- shown that male steriles occur on the X and autosomes tors could lead to an underestimate of the frequency roughly in proportion to theireuchromatic lengths of female sterility factors. However, it is very unlikely (LINDSLEYand LIFSCHYI-z1972). (2) The distributions to result in a substantial bias in the female us. male of homozygous and hemizygous effects of individual comparison because the breeding scheme provides lit- sterility factors are similar. In addition, it should be tle opportunity for differential selection of male and emphasized that the difference reported here is tenta- female factors because they are in complete linkage tive (because of the assumption of equalsegment disequilibrium within each introgression subline. Selec- length distributions on the X and autosomes) and may tion must operate by changing the frequencies of intro- Hybrid Incompatibility Hybrid in Drosophila 831

gressed segments of different lengths. If female sterility generally accumulate more rapidly than female factors factors are selected against when heterozygous, smaller in Drosophila species. segments would be favored. However, smaller segments It is not clear whether differential accumulation of are also less likely to contain male sterility factors. If male and female factors is due to a higherrate of evolu- male and female factors areinterspersed along the tion on a per locus basis or whether more loci poten- chromosome, female sterility factors cannot be elimi- tiallyaffect male than female gametogenesis inDro- nated without also eliminating male factors at nearly sophila. In one EMS mutagenesis experiment in D. the same rate. In any case, the data on segment length melanogarter, significantly more male than female ster- discussed earlier suggests that, if anything, selection has iles wererecovered on the autosomes (231 male specific favored longer introgressions due to their heterotic ef- and 140 female specific; LINDSLEYand LIFSCHWZ1972), fects. In addition, the second chromosome introgres- whereas two P-element mutagenesis experiments gave sions of H. HOLLOCHERand C.4 WU (personal commu- the opposite result (38 malespecific and 75 female nication) also confer greater male than female sterility specific; CASTRILLON et al. 1993; COOLEXet al. 1988). even though, in theirdesign, repeated backcrosses were WU and DAVIS(1993) have suggested three possible made with hybrid males rather than hybrid females. causes of a morerapid rate of male than female sterility The high frequency of male sterile introgressions on factors: the Xprevents an assessment of the corresponding fre- 1. X-linked recessive or partially recessive mutations af- quency of X-linked female steriles in this experiment fecting male fitness will have a greater rate of substi- (although none were detected among the male fertile tution than autosomal mutations, whereas no such lines). However, the attached-X experiment of COYNE difference is expected for mutations that affect only (1985) shows that when the entire X of simulans is ho- female fitness (CHARLESWORTHet al. 1987). This re- mozygous in a simulans/mauritiana or simuLans/secheLlia sult can account for a difference in frequency of hybrid background, thefemales are fully fertile whereas male and female hybridsterility factors on the X the corresponding F1 males are sterile. TURELLIand chromosome (COYNEand Om 1989a), but not on ORR(1995) suggest that this result does not necessarily the autosomes. So, this theory might help explain mean that male sterility factors accumulate more rap- the apparent excess of male sterility factors on the idly than female factors, because the Xcould bedevoid X chromosome, but it cannot explain the marked of female factors by chance if the genome contains a excess on the autosomes. small number of both male and female factors. How- 2. Sexual selection for male reproductive characters ever, this situation seems unlikely because genetic map- may result in the rapidevolution of genes that confer ping experiments indicate that the Xchromosomes of sterility in hybrids. The main evidence is that closely simulans, mauritiana and sechellia contain numerous fac- related species of animals frequently differ more in tors thatcontribute to malesterility in hybrids (re- male reproductive characters than in other types of viewed by WU and PALOPOLI1994). characters. Backcross experiments involving other pairs of Dro- 3. Some special properties of spermatogenesis in heter- sophila species also indicate that autosomal male steril- ogametic male animals such as Drosophila and mam- ity factors accumulate more rapidly than female factors. mals might make this developmental system particu- In these experiments, females of genotype X'/X'; A'/ larly susceptible to perturbation inhybrids. One AI or 2 can be compared with males of equivalent geno- such property is that little or notranscription occurs type, XI/ Y; A'/A' Or * (where A refers to all autosomes after meiosis andduring spermiogenesis (FULLER and superscripts refer to species 1 and 2). Among seven 1993). Another is the male-specific sterility of X-au- such comparisons, four show essentially full fertility of tosome translocations, which is probably related to both males and females, while three show significantly greater female than male fertility. The four with full the precocious inactivation of the X in primary fertility of both sexes are D. virilis with texana, amm'cana, spermatocytes (LIFSCHWZand LINDSLEY1972). lummei, and novamexicana ( ORRand COYNE1989). In a More work is needed to verify that a relatively rapid persimilis/pseudoobscura backcross, 57% ofmales and rate of evolution of male sterility factors is characteristic 82% of females of the above genotypes were fertile of Drosophila species and to determine whether it oc- (Om1987); in a pseudoobscura Bogota/USA backcross, curs in other types of organisms. It is particularly im- 87% of males and 99% of females were fertile (Om portant to know whether male factors evolve rapidly in 1989); and in a virilas/littoralis backcross, 54% of males female-heterogametic species such as lepidopterans and 83% of females were fertile (Omand COYNE1989). and birds. A comparison between male- and female- Thus, there are at least three phylogenetically inde- heterogametic species may provide further insight into pendent hybrids that show evidence of a greater male the mechanisms. For example, if male sterility factors than female sterility in comparable genotypes (those do not evolve rapidly in lepidopterans whereas they do involving D. simulans, virilis and pseudoobscura). These in Dipterans, the sexual selection hypothesis would be results support the hypothesis that male sterility factors called into question, because both groups show much 832 J. R. True, B. S. Weir and C. C. Laurie the same evidence of sexual selection in terms of rapid If T > 1 (greater male than female effects), recessivity divergence of male secondary sexual characters. is not necessary for obtaininghybrid fitnesses that favor Inviability factors: The frequency of homozygous le- HALDANE’Srule. As noted by TURELLIand Om, R can thal introgressions is similar to the frequency of female be greater than 1 even for fully dominant factors. For steriles and both are much less than the frequency of example, T need only be greater than 1.22 to make R male steriles. These differences probably reflect a differ- > 1 when d = 1.0 and p, = 0.36 (as in D. simulans). ence in evolutionary rate since lethal mutations occur Thus, it appears thatwhen effects on theheterogametic at 10-15 times the rateof steriles in mutagenesis experi- sex evolve substantially more rapidly than effects on ments, as noted above. Several other studies (summa- the homogametic sex, as observed in this study of two rized by WU and DAWS1993) have also provided indica- Drosophila species, then hybrid fitnesses favor HAL- tions that inviability factors accumulate more slowly DANE’S rule, regardless of the degree of dominance. than male sterility factors in Drosophila. For example, If T < 1, hybrid fitnesses that favor HALDANE’S rule the Drosophila species pairs hydei/neohy&i and moja- can only be obtained if the incompatibility factors are uensis/arizonae show much stronger effects on male ste- very recessive. For example, if r = 0.50 and p, = 0.36, rility than on viability in backcross experiments (HEN- then d must be less than 0.13 for R to be greater than NIG 1977, ZOUROS 1981, 1989). 1. The required d is 0.03 when p, = 0.06, as for a typical ‘The TURELLIand Om composite model: In the fol- lepidopteran with 31 equally sized chromosomes (ROB- lowing discussion, “male” refers to the heterogametic INSON 1971, p. 88). This situation is relevant to how sex and “female” to the homogametic sex, unless noted HALDANE’S rule applies to birds and lepidopterans in otherwise. which malesare homogametic (ZZ) and females hetero- In general, the relative levels of fitness in male and gametic (2“). If male (ZZ) fertility factors evolve more female hybrids may depend on the degree of domi- rapidly than female (ZW) factors, then r < 1, and in- nance of incompatibility factors, the relative numbers compatibility factors must be almost completely reces- of male us. female specific factors and the relative distri- sive on the average to get the high degree of compli- bution of these factors on theX us. autosomes. A simple ance to HALDANE’S rule that is observed in these groups. model of hybrid fitness has been developed by TURELLI Such a low level of recessivity seems possible because and ORR (1995, Appendix B), which is very useful in lethal mutations in D. melunogaster reduce the viability assessing the relative importance of these factors. In of heterozygotes by only 2-6% (SIMMONSand CROW this model, T representsthe ratio of the cumulative 1977). Empirical data are clearly needed to settle this effectsof incompatibilities affecting hybrid male to issue, as noted earlier. those affecting hybrid female fitness (due either to dif- The quantity p, is the proportion of incompatibilities ferent numbers of genes or to differences in their ho- involving X-linked loci, which is related to the “X ef- mozygous/hemizygous effects), p, represents the frac- fect” discussed earlier. The effect of p, on R depends tion of incompatibilities involving X-linked loci and d on the degree of dominance. For partially recessive in- is the degree of dominance (assumed here to be equal compatibility factors, increasing p, favors the occur- for males and females). Equations B1 of TURRELLIand rence of HALDANE’S rule,while for partially dominant ORRgive R, the ratio of hybrid breakdown scores for factors, increasing p, has the opposite effect. Males suf- males and females. HALDANE’S rule is favored when R fer less from X-linked dominants relativeto females > 1 (i.e., when hybrid males suffer more hybrid break- because females have two X chromosomes and there- down than hybrid females): fore twiceas many X-linked incompatibility factors, whereas they suffer relatively more from X-linked reces- 7[px/2 + (1 - R= sives because the effects in females aresheltered by d heterozygosity. TURELLI andORR note that, if 7 = 1 (ie., no differ- Possible causes of HALBANE’S de: HALDANE’Srule ence in number oreffects of male and female factors), that heterogametic hybrids are more adversely affected a sufficient condition for HALDANE’S rule is that the thanhomogametic hybrids cuts across phylogenetic incompatibility factors are recessive or partially reces- boundaries (birds, mammals, flies and lepidopterans) sive (d < irrespective ofhow the effects are allo- and applies to both viability and fertility (although the cated between the X and autosomes (provided that $I, viability pattern is not as clear for male heterogametic > 0). In the case of inviability, factors with sex-specific species as it is for female heterogametic species). The effects are expected to be very rare, because most cases generality of this pattern suggests a commonunderlying of lethalmutations in Drosophila affect both sexes. cause, but this may not be thecase. In fact, the evidence Therefore, most examples of HALDANE’Srule for viabil- to date strongly indicates that HALDANE’S rule has at ity probably result from the recessivity ofhybrid effects. least two causes: recessivity of incompatibility factors The attached-X experiments of ORR (1993b), which and differential accumulation of sterility factors affect- produce“unbalanced” females, provide strong evi- ing males and females [as suggested by ORR (1993b) dence that this is true for two Drosophila species pairs. and WU and DAVIS (1993)l. Hybrid Incompatibility in Drosophila 833

As noted earlier, the attached-Xexperiment of ORR We are most grateful to SAMANTHAGASSON for her careful and patient work on the difficultjobof constructing and maintaining the (1993b) provides strong evidence that inviability factors introgression lines, to LYNN STAMfor the molecular marker analysis are recessive or partially recessive in hybridizations in- and to MERYL CARTERand ROBERTWARD for additional technical volving species of the melanogaster subgroup of Dro- assistance. Very helpful reviews of the manuscript were provided by sophila. Because there is no reason to suspect differen- J. COYNE,B. CHARLESWORTHand M. TURELLI.We also thank C.4 tial accumulation of male us. female factors affecting Wu, H.HOI.I.OCHER, H. A. ORRand M. TUREI.LIfor providing copies of their papers prior to publication. Thiswork was supported by U.S. = hybrid inviability, we assume that T 1 in the TURELLI Public Health Service Grants GM-47292 and GM-45344. and ORRmodel, which means that partial recessivity is both necessary and sufficient to account forHALDANE’S rule. Therefore,partial recessivity clearly explains HAL LITERATURE CITED DANE’S rule forinviability in some Drosophila hybridiza- ASHBURNER, M., 1989 Drosophi1a:A Laboratory Handbook. Cold Spring tions and also can provide a general explanationof the Harbor Press, Cold Spring Harbor, NY. BAKER, B.S., 1973 The maternal andzygotic control ofdevelopment inviability results in both male and female heteroga- by cinnamon, a new mutant of Drosqbhila melanogaster. Dev. Biol. metic groups. 33: 429-440. In contrast to the inviability results, there is no strong BIER,E., H. V’AESSIN,S. SHEPHERD,K. LEE, K. MCCALL et al., 1989 Searching for pattern and mutation in the Drosophila genome evidence that sterility factors are partially recessive in with a P-1acZvector. Genes Dev. 3: 1273-1287. Drosophila (or any other group). Moreover, their de- BOCK,I. R., 1984 Interspecific hybridization in the genus Drosophila. gree of dominancemay be irrelevant to theobservation Evol. Biol. 18: 41-70. CARIOU,M.-L., 1987 Biochemical phylogeny of the eight species in of HALDANE’S rule in some hybridizations, such as that the Drosophila melanogaster subgroup, including D. sechellia and between D. simulans and mauritiana. In this case, the D. orena. Genet. Res. 50: 181-185. CASTRILLON,D. H., P. GONCZY,S. ALEXANDER,R. RAWSON, C. G. attached-Xexperiment of COYNE(1985) shows that fe- ERERHAKTet al., 1993 Toward a molecular genetic analysis of male hybrids are fertile (while males are sterile) not spermatogenesis in Drosophilamelanogusterr characterization of because their X-linked sterility factors are recessive, but male-sterile mutants generated by single Pelement mutagenesis. Genetics 135: 489-505. rather because they either have no X-linked female ste- CHARLESWORTH,B., J. A. COYNEand N. H. BARTON,1987 The rela- rility factors or they have an insufficient number to tive rates of evolution of sex chromosomes and autosomes. Am. produce sterility even when those factors are homozy- Nat. 130: 113-146. CHARLESWORTH,B., J. A. COYNEand H. A. ORR,1993 Meiotic drive gous. Therefore, recessivity (even if it exists, which is and unisexual hybrid sterility: a comment. Genetics 133: 421- not unlikely) cannot explain HALDANE’Srule in this 424. hybridization. The results of the introgression experi- COOLEY,L., R. KFLLEY and A. SPRADLING,1988 Insertional mutagen- esis of the Drosophila genome with single P elements. Science ment reported here strongly indicate that HALDANE’S 239: 1121-1128. rule is observed in this hybridization because there are COYNF.,J. A,, 1984 Genetic basis of male sterility in hybrids between many more male than female sterility factors. In this two closely related species of Drosqbhila. Proc. Natl. Acad. Sci. USA 81: 4444-4447. situation (T > 1 with male heterogamety), HALDANE’S COYNE,J. A,, 1985 The genetic basis of Haldane’s rule. Nature 314 rule is favored even if sterility factors are completely 736-738. dominant (TURELLIand ORR1995). COYNE,J. A,, 1989 Genetics of sexual isolation between two sibling species, Drosophila simulans and Drosophila mauritiana. Proc. Natl. Although a more rapid rateof male than female ste- Acad. Sci. USA 86: 5464-5468. rility factors can explain HALDANE’S rule inmale-hetero- COYNE,J. A., 1992 Genetics and speciation. Nature 355: 511-515. COYNE,J. A. and H. A. ORR,1989a Patterns of speciation in Drosoph- gametic species (and very likely does in Drosophila), ila. Evolution 43: 362-381. this process works against HALDANE’Srule in female- COYNE,J. A. and H. A. ORR,1989b Two rules of speciation, pp. heterogametic species. Nevertheless it is possible to get 180-207 in Speciation and Its Cons~quences,edited by D. OTTKand J. A. ENDLER.Sinauer Associates, Sunderland, MA. (T HALDANE’Srule under this condition < 1 with female FISHEK,R. A., 1949 The Theory of Inbreeding. Hafner, New York. heterogamety), but the sterility factors must be very FOSS, E., R. LANDP, F. W. STAHLand C. M. STEINBERG,1993 Chiasma recessive (TURELLIand ORR1995). However, the only interference as a function of genetic distance. Genetics 133: 681- 691. way that the differential accumulation hypothesis could FULLER,M. T., 1993 Spermatogenesis, pp. 71-148 in TheDeuelopment provide a general explanation of HALDANE’Srule is if of Drosophila melanogaster, edited by M. BATEand A. MARTINEL- heterogametic (not specifically male) sterility evolves ARI&. Cold Spring Harbor Laboratory Press, Cold Spring Har- bor, NY. more rapidly thanhomogametic sterility. Unfortu- HALDANE, J. B. S., 1922 Sex-ratio and unisexual sterility in hybrid nately, there is no evidence regarding the relative num- animals. J. Genet. 12: 101-109. bers of male and female factors in female heteroga- HENNIG,W., 1977 Gene interactions in germ differentiation of Dro- sophila, pp. 363-371 in Advances in Enzyme Regulation, edited by metic species. G. WEBER.Permagon, Oxford. We conclude that bothrecessivity of inviabilityfactors HEY,J., and R. M. KLIMAN, 1993 Population genetics and phyloge- and the differential accumulation of male and female netics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex. Mol. Biol. Evol. 10: sterility factors play important roles in the observation 804-822. of HALDANE’S rule. Further work is needed to explore JOHNSON,N. A., and C:I. WU, 1993 Evolution of postmating reprw ductive isolation: measuring the fitness effects of chromosomal their relative importance, especially in female heteroga- regions containing hybrid male sterility factors. Am. Nat. 142: metic species. 213-223. 834 J. R. True, B. andS. Weir C. C. Laurie

JOHNSON,N. A., D. E. PEREZ,E. L. CABOT,H. HOLLOCHERand C.-I. ZOUROS,E., 1981 The chromosomal basis ofviability in interspecific WU, 1992 A test of reciprocal X-Y interactions as a cause of hybrids between Drosophila arizonasis and Drosophila mojavensis. hybrid sterility in Drosophila. Nature 358: 751 -753. Can. J. Genet. Cytol. 23: 65-72. LACHAISE, D., M.-L. CARIOU, J. R. DAVID,F. LEMEUNIER,L. TSACAS et ZOUROS,E., 1989 Advances in the genetics of reproductive isolation al., 1988 Historical biogeography of the Drosophila melanogastm in Drosophila. Genome 31: 211-220. species subgroup. Evol. Biol. 22: 159-225. LEMEUNIER,F., and M. ASHBURNER, 1976 Studies on the evolution Communicating editor: A. G. CIARK of the melanogester species subgroup of the genus Drosophila (So- phophora). 11. Phylogenetic relationshipsof six species based upon polytene chromosomebanding patterns.Proc. R. Soc. APPENDIX: THELENGTH OF INTROGRESSED Lond. B Biol. Sci. 193: 275-294. SEGMENTS LIFSCHYTZ,E., and D. L. LINDSLEY,1972 Sex chromosome activation during spermatogenesis. Genetics 78: 323-331. Here we address the question of how much of the LINDSLEY,D. L., and E. LIFSCHYTZ,1972 The geneticcontrol of mauritiana genome remains associated with the selected spermatogenesis in Drosophila, pp. 203-222 in Proctedings of the International Symposium on the Genetics of the Spermntozoon, edited P elementmarker after 15 generations of repeated by R. A. BEATIY andS. GILJECKSOHN-WAELSCH.University of Ed- backcrossing to simulans. The experimental design is a inburgh, Edinburgh. standard one, and there is some simple theory that LINDSIXY,D. L., and K T. TOKUYASU,1980 Spermatogenesis, pp. 226-294 in The Genetics and Biology of Drosophila, edited byM. addresses the distribution of the length of the intact ASHBURNER and T. R. F. WRIGHT.Academic Press, New York. segment associated with a marker after t generations. LIU,J.,J.M. MERCER,L. F. STAM, G. GIBSONand C. C. LAURIE,1996 FISHER(1949) showed that the probability density for Genetic analysis of a morphological shape difference in the male genitalia of Drosophila simulans and D. mauritiana. Genetics 142: a length of x Morgans on each side of the marker is (4) (in press). f(x) = te-lX,which has a mean of l/t and a variance of MULLER,H. J., 1940 Bearing of the Drosophila work on systematics, l/t2. Recently, NAVEIRA and BARBADILLA (1992) pp. 185-268 in The New Systematics, edited by J. HUXLEY. Clarendon Press, Oxford. pointed out that Fisher’s derivation of the mean and NAVEIRA,H., and A. BARBADILIA, 1992 The theoretical distribution variance assume an infinitely long chromosome. They of lengths of intact chromosome segments around a locus held provide exact results for afinite chromosome and show heterozygous with backcrossing in adiploid species. Genetics 130: 205-209. that an estimated mean of (l/t)is reasonably accurate NAVEIRA,H. F., 1992 Location of X-linked polygenic effects causing after the first several generations unless the marker is sterility in male hybrids of Drosophila simulans and D. mauritiana. close to a telomere. Heredity 68: 211-217. Om, H. A., 1987 Genetics of male and female sterility in hybrids Fisher’s derivation also assumes a Poisson distribu- of Drosophila pseudoobscura and D. persimilis. Genetics 116: 555- tion of crossovers (ie., no interference), which is not 563. justified in Drosophila (ASHBURNER 1989). Here we pro- Om, H. A,, 1989 Genetics of sterility in hybrids between two subspe- vide numerical results for the mean and distribution of cies of Drosophila. Evolution 43 180-189. Om, H. A,, 1993a A mathematical model of Haldane’s rule. Evolu- the intact segment length when there is interference tion 47: 1606-1611. of the type modeled by Foss et al. (1993), which fits Om, H. A., 1993b Haldane’s rule has multiple genetic causes. Na- multipoint recombination data from Drosophila very ture 361: 532-533. Om, H. A,, and J. A. COYNE,1989 The genetics of postzygotic isola- well. tion in the Drosophila uirilis group. Genetics 121: 527-537. In general, the probability density for length x of ROBERTSON,H. M., 1988 Mating asymmetries and phylogeny in the intact segmentis g( = t P‘”, where Pis the probability Drosophila melanogaster species complex. Pac. Sci. 42: 72-80. x) ROBINSON,R., 1971 Lepidoptera Genetics. Permagon Press, Oxford. of no crossovers in x within a tetrad. Under the FOSSet SIMMONS,M. J., and J. F. CROW,1977 Mutations affecting fitness in al. (1993) model of crossing over, P is given by (Al) Drosophila populations. Annu. Rev. Genet. 11: 49-78. where m = 4 for Drosophila. STUART,A,, and J. K. Om, 1987 Kendall’s Aduanced Theq of Statistics. Volume I. Distribution Themy. Oxford Univ. Press, Oxford. TRUE,J. R., J. M. MERCERand C. C. LAURIE, 1996 Differences in crossover frequency and distribution among threesibling species of Drosophila. Genetics 142: 507-523. TUREILI,M., and H. A. ORR, 1995 The dominance theory of HAL Numerical integration with t = 15 gives a mean value DANE’S rule. Genetics 140: 389-402. VIGNEAUI.T,G., and E. ZOUROS,1986 The genetics of asymmetrical of 0.047 for the distributionof intact lengths, compared male sterility in Drosophila mojavensis and Drosophila arizonensis with a value of l/t = 0.067 assuming no interference. hybrids: interactions between the Y-chromosome and autosomes. Figure 1 shows the shape of the distribution (allowing Evolution 40: 1160-1 170. Wu, C.-I., and A. T. BECKENBACH,1983 Evidence for extensive ge- for interference), which is very asymmetric. netic differentiation between the sex-ratio and the standard ar- To determine experimentally the length of the intact rangement of Drosophilapseudoobscura and D.persimilis and identi- segment associated with a P marker would be very diffi- fication of hybrid sterility factors. Genetics 105 71-86. Wu, C.-I., and A. W. DAVIS,1993 Evolution of postmating reproduc- cult, butinferences about the mean lengthcan be made tive isolation: the composite nature of Haldane’s rule and its by sampling a molecular marker locus in a number of genetic bases. Am. Nat. 142: 187-212. different introgression lines and asking whether it has Wu, C.-I., and M. F. PALOPOLI,1994 Genetics of postmating re- productive isolation in animals. Annu. Rev. Genet. 27: 283- the mauritiana or simulans allele. This was done for five 308. loci (three on the third, one on the second and one ZENG, L.-W., and R. S. SINGH,1993 The genetic basis of HALDANE’s on theXchromosome) , by scoring a restriction site that rule and the nature of asymmetric hybrid male sterility among Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. differs between the mauritiana and simulans stocks used Genetics 134: 251-260. in the experiment. Hybrid Incompatibility in Drosophila 835

125 jan (99E)

030

0 ksul

0 mauritiana allele

(xTa3 03 o simulans allele 03 ...... ka.26 0 """" k=l .OO

...... ~ ...... " ...... 25]- I I , _I"""""""""""". f 0 0 0 w x 3 subllines inrank order by map distance

sli (52D)

ooocm 0 0- 8 03 5 -i 03 3 20 ...... o""'"""."'.'." ....." ...... I...... e...... I......

fpf "- O0""""""""""""""" -" -" "_" - "" """"""_ """"""""""""""""" 40: 40: 0"I 1 I I I I I I I 0 El w x i3 s 2 w x i sublines in rank order by map distance sublines in rank order by map distance I 5 030oooo- 9 030 a, E oooo i2 40 030 0 cuOo aa

...... I.... ".." ...... ~ ...... " ...... 20 "...... -...... m-_""_"""""""""" b 0 y-" I I I 0-0"""""""""""""""""~ 0 0 w x 3 s2 kk"-l2 w x 3 sublinesin rank order by map distance sublines in rank order by map distance

FIGUREA1.-Molecular marker analysis of introgression sublines. Each point in a plot represents a single subline and the symbol indicates whether it has the maun'tiana or simulans allele at the molecular marker locus. The ordinate represents the genetic map distance (centiMorgans) between the Pinsert within a subline and the molecular marker locus, based on mapping data from a pure mauritiana strain. If the introgressed segment in a subline is longer than the distance between its P element and the molecular marker, it will have the maun'tiana allele; otherwise it will have the simulans allele. Thus, the ordinate for black (maun'tiana) symbols represents a lower limit for the length of the introgressed segment within that line and the white (simulans) segments represent an upper limit. For reference, the dashed lines represent the expected mean length of an introgressed segment if k = 1 (i.e., if hybrids during the introgression show the same level of recombination as pure maun'tiana) or k = 0.264 (i.e., if hybrids show only 0.264 times as much recombination as pure mauritiana).

Interpretation of the molecular marker results re- The position of each molecular marker on this map quires estimates of themap distance between the was estimated by interpolation between cytologically marker and each of the P inserts analyzed. A complete flanking P inserts and assuming a constant coefficient genetic map of the mauritiana genome has been con- of exchange within that interval. Figure A1 confirms, structed by estimating the recombination fraction be- in a qualitative way, the obvious expectation that an tween each pair of adjacent Pinserts (TRUEet al. 1996). introgression line is less likely to contain the mauritiana 836 J. R. True, B. S. Weir and C. C. Laurie

TABLE A1 Molecular marker analysis of introgression lines

Significance level" Molecular No. P No. sublines MLE" Log marker positions analyzed k k = 1.000 k = 0.264 (W' jun (99E) 15 42 0.181 < 10"; 0.708 17.1 h (66D) 12 27 0.224

" MLE = maximum likelihood estimate of k. "Significance level given either k = 1.000 or k = 0.264. ' Log(LR) = Log of ratio of likelihood of k = 0.264 to likelihood of k = 1.000. '' k for sli could not be estimated because all sublines had the simuluns allele. Fifteen of the same sublines were analyzed for both jan and Antp. allele at the molecular marker locus, as map distance tiana flies. The other estimate (for Antp) is 1.1 1, sug- between the P insert and the molecular marker locus gesting essentially the same level of recombination as increases. However, the segment lengths appear longer in mauritiana flies. than expected, based on a predicted mean of 0.047, so To determine whether the common value of k = in the following analysiswe consider the possibility that 0.26 provides a satisfactory fit to the data for each recombination within the introgression lines differs molecularmarker, a significance level was deter- from that within pure mauritiana flies by a constant mined by calculating L for all possible outcomes of factor, k. the experiment for that particular marker (i.e., all The probability that the mauritiana allele at a marker possible sets of values of ag and 6, with the same locus remains on the same chromosome with the se- sums) and then summing all the probabilities less lected P insert after t generations of backcrossing is than or equal to theprobability for theobserved out- Q(x), where r is the recombination fraction between come. The results in Table 1 show that k = 0.26 pro- the two loci within mauritiana, x is the corresponding vides an adequate fit for all markers, even Antp. In map distance, and k is a factor to be estimated. The contrast, the significance level values with k = 1 are quantity kr represents the effective level of recombina- very low for jan, sli and run (