IT is well established that neutral variation is affected Drosophila melanogaster (Charlesworth et al. 1993; Hudby

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1 Copyright 2002 by the Genetics Society of Americ Chromosoml Ptterns of Microstellite Vribility Contrst Shrply in Africn nd Non-Africn Popultions of Drosophil melnogster M. Kuer, B. Zngerl, D. Dieringer nd C. Schlötterer 1 Institut für Tierzucht und Genetik, 1210 Wien, Austri Mnuscript received June 11, 2001 Accepted for publiction October 29, 2001 ABSTRACT Levels of neutrl vrition re influenced by bckground selection nd hitchhiking. The reltive contribution of these evolutionry forces to the distribution of neutrl vrition is still the subject of ongoing debtes. Using 133 microstellites, we determined levels of vribility on X chromosomes nd utosomes in Africn nd non-africn D. melnogster popultions. In the ncestrl Africn popultions microstellite vribility ws higher on X chromosomes thn on utosomes. In non-africn popultions X-linked polymorphism is significntly more reduced thn utosoml vrition. In non-africn popultions we observed significnt positive correltion between X chromosoml polymorphism nd recombintion rte. These results re consistent with the interprettion tht bckground selection shpes levels of neutrl vribility in the ncestrl popultions, while the pttern in derived popultions is determined by multiple selective sweeps during the coloniztion process. Further reserch, however, is required to investigte the influence of inversion polymorphisms nd unequl sex rtios. IT is well estblished tht neutrl vrition is ffected Drosophil melnogster (Chrlesworth et l. 1993; Hudby selection t linked sites. Two contrsting modes of son 1994). A good fit to the dt, however, ws lso obselection, bckground selection nd hitchhiking, hve served for simple hitchhiking model (Wiehe nd Steprimrily been investigted. The hitchhiking model s- phn 1993; Stephn 1995). Kim nd Stephn (2000) sumes tht recurrent beneficil muttions re spreding nlyzed joint effects of hitchhiking nd bckground through the popultion nd tht linked neutrl vrints selection on neutrl vrition nd found tht sttionry become fixed in ssocition with the beneficil lleles levels of nucleotide vribility in low recombining re- (Mynrd Smith nd High 1974; Kpln et l. 1989). gions re influenced minly by hitchhiking, wheres The lterntive model of bckground selection is bsed in regions with higher recombintion rte bckground on the continuous removl of deleterious muttions selection hs more impct (Kim nd Stephn 2000). from popultion (Chrlesworth et l. 1993). Inter- Approches to distinguish between bckground seestingly, both models mke similr predictions for levels lection nd hitchhiking: Three pproches hve been of neutrl vribility in regions of different recombin- proposed to discriminte between the reltive roles of tion rtes. Neutrl vrition in regions of low recombi- bckground selection nd hitchhiking in shping gention is more suppressed thn in regions of high re- nome-wide vribility. A summry of the expecttions combintion, leding to positive correltion between under the different pproches is given in Tble 1. recombintion rte nd vribility (Mynrd Smith First, t muttion-selection equilibrium, the two modnd High 1974; Kpln et l. 1989; Chrlesworth et l. els differ in their predictions for the single nucleotide 1993). Experimentl dt for rnge of different species polymorphism (SNP)-frequency spectrum in genomic demonstrted tht this predicted pttern is widespred regions of low recombintion. The hitchhiking model phenomenon (Stephn nd Lngley 1989, 1998; Nch- predicts surplus of rre lleles compred to neutrl exmn 1997; Nchmn et l. 1998). The qulittive simi- pecttions for equilibrium popultions (Brvermn et lrity of both models hs mde it difficult to determine l. 1995), while under the bckground selection model the reltive importnce of bckground selection nd in lrge popultions only slight skew in the llele frehitchhiking in shping the genomic distribution of neuquency distribution is expected (Hudson nd Kpln trl vrition. Recently, it hs been shown for set of 1994). Empiricl dt, however, did not lwys detect resonble prmeters for recombintion nd deleteristtisticlly significnt skew towrd rre lleles in the ous muttion rte tht the bckground selection model llele frequency distribution (Agudé et l. 1989; Mrprovides good fit to the levels of vrition observed in tin-cmpos et l. 1992; Begun nd Aqudro 1995). Finlly, in recent study, Fy nd Wu (2000) found tht 1 n excess of derived high frequency lleles is lso Corresponding uthor: Institut für Tierzucht und Genetik, Josef Buunique chrcteristic of selective sweeps. mnn Gsse 1, 1210 Wien, Austri. E-mil: christin.schloetterer@vu-wien.c.t A second pproch utilizes the comprison of mrk- Genetics 160: ( Jnury 2002)

2 248 M. Kuer et l. TABLE 1 lleles with the deleterious muttion. Given tht deleterious Expecttions under different pproches to distinguish lleles nd their linked neutrl vrition re purged hitchhiking nd bckground selection from the popultion, the bckground selection model Hitch- Bckground predicts more neutrl vrition on the X chromosome thn on the utosomes fter correction for different Expecttion hiking selection popultion sizes of the chromosomes nd ssuming Excess of rre lleles 1:1 sex rtio. It should be noted, however, tht this does Excess of derived high frequency not necessrily imply tht X chromosomes hve higher lleles bsolute level of polymorphism. Correltion of vribility with Similrly, due to the mle heterogmy, beneficil murecombintion rte ttions hve longer sojourn time on utosomes thn Mrkers with very high on the X chromosome. In contrst to the bckground muttion rte selection model, under which deleterious muttions re Mrkers with low muttion rte removed from the popultion, the hitchhiking model Higher reltive X chromosoml ssumes the fixtion of beneficil muttions. It hs been vribility shown tht beneficil muttions on the X chromosome Lower reltive X chromosoml hve higher fixtion rte thn utosomes if selection vribility opertes on new muttions (Chrlesworth et l. 1987). Under these ssumptions the hitchhiking model, therefore, predicts more neutrl vrition on the utosomes ers with different muttion rtes such s sequence polymorphism thn on the X chromosome (fter correction for the dif- nd microstellites (Schug et l. 1998). The ferent popultion sizes of the chromosomes nd ssum- expecttion is tht vribility, s mesured with mrkers ing 1:1 sex rtio). Recently, Begun nd Whitley (2000) with high muttion rtes, should show correltion with followed this pproch nd found X chromosomes to hrbor recombintion rte under bckground selection but not less sequence vrition thn utosomes in D. simulns. under hitchhiking. Mrkers with low muttion rtes In this report we focus minly on the third pproch. should show correltion in both cses. Recently, levels We used microstellites, which re neutrl polymorphic of microstellite vribility were correlted with recom- mrkers (Schlötterer 2000), to determine levels of bintion rtes to discriminte between bckground selection neutrl vribility on the X chromosome nd utosomes nd hitchhiking for D. melnogster (Michlkis in popultions from Afric nd non-africn popul- nd Veuille 1996; Schlötterer et l. 1997; Schug et tions. The comprison between the two groups of popu- l. 1998). The obtined results were not consistent, due ltions ws motivted by the coloniztion history of D. lrgely to the reltively smll number of loci nlyzed melnogster. It is well estblished tht the species origi- (Schlötterer 2000). Furthermore, the reltively low nted in Afric nd expnded its hbitt only recently, muttion rte of D. melnogster microstellites (Schlötterer 10,000 yers go (Dvid nd Cpy 1988). Therefore et l. 1998; Schug et l. 1998b) limits their power it seemed very likely tht different evolutionry forces to discriminte between bckground selection nd selective were opertive in these groups nd more detiled infor- sweeps. Humn microstellites, however, hve mtion cn be glened if these groups re nlyzed significntly higher muttion rte thn D. melnogster seprtely. Africn popultions re presumbly closer microstellites, but no correltion between microstel- to n equilibrium stte thn non-africn popultions, lite vribility nd recombintion rte ws detected which in turn could be influenced by founder events, (Pyseur nd Nchmn 2000). bottlenecks, nd fixtion of beneficil muttions ssocited The third pproch to distinguish between the two with the coloniztion process. selection models ws proposed by Aqudro et l. (1994) We show tht X chromosomes hrbor significntly nd concentrtes on different vribility levels of X chromosomes more vrition thn utosomes in Africn popultions nd utosomes. Under certin ssumptions but not in non-africn popultions. The reduction in the models differ in their predictions bout the levels of vribility of non-africn compred to Africn X chro- sequence vrition on X chromosomes nd utosomes mosomes ws found to be more pronounced in regions (Chrlesworth et l. 1993). In most species mles of low recombintion rte. We discuss the two selection crry single X chromosome nd two utosomes, while models nd other evolutionry forces such s inversion femles hve two copies of both. In such heterogmetic polymorphism nd unequl sex rtios in the light of system recessive deleterious muttions re purged our dt. more efficiently from the popultion when locted on the X chromosome (Crow nd Kimur 1970; Lngley et l. 1981). Becuse deleterious lleles re removed MATERIALS AND METHODS more rpidly from the X chromosome there is less Microstellites: We nlyzed 133 microstellite loci of which chnce for recombintion to combine different neutrl 40 loci re locted on the X chromosome nd 93 on the

3 Microstellite Vribility in D. melnogster 249 It should be noted tht Equtions 2 nd 3 ssume stepwise muttion model (Oht nd Kimur 1973). In mmmls mles nd femles hve different muttion rtes (Bohossin et l. 2000), which would require n djust- ment for the observed level of vribility becuse then X chromosomes nd utosomes would hve different muttion rtes. A recent study in D. melnogster, however, filed to demonstrte significnt effect for bse substitutions (Buer nd Aqudro 1997); therefore, we lso ssumed no systemtic bis in microstellite muttion rtes mong loci locted on X chromosomes nd utosomes. Recombintion rtes for ll loci were clculted s outlined in Comeron et l. (1999). Units of recombintion given in this rticle re recombintion events/(bse pir gener- tion). The lck of recombintion in Drosophil mles ws ccounted for by correction fctor of 2 3 for X chromosomes nd 1 2 for utosoml loci. Furthermore we controlled whether, for X chromosomes nd utosomes, n equl frction of loci re locted in regions of similr recombintion rte (fter djustment for no recombintion in mles). X chromosoml nd utosoml loci, included in this study, therefore experienced on verge similr rte of crossing over (P 0.05, Mnn Whitney U-test). utosomes. The selected microstellite loci re locted in chromosoml regions covering wide rnge of recombintion rtes. All loci were typed in Africn nd non-africn popultions. The dt for 28 loci were tken from the literture (Bchtrog et l. 2000). Primer sequences, nneling tempertures, repet mo- tifs, nd cytologicl positions of ll loci re vilble from the uthors homepge ( Micro- stellite nlysis followed stndrd protocols (Schlötterer nd Zngerl 1999). Fly stocks: Africn isofemle lines were from Zimbbwe or Keny or from both popultions. Depending on the locus, flies were typed from these popultions. To ccount for inbreeding effects during propgtion of these lines we rndomly selected one llele in heterozygous individuls. Non-Africn smples were F 1 flies from Austri, Frnce, Ger- mny, Itly, the Netherlnds, Russi, nd the United Sttes. Twenty-five to 30 individuls per popultion were typed. Not ll loci were typed for the complete set of popultions. The minimum popultion smple t locus consisted of 25 Africn nd 60 non-africn individuls. If more thn single popultion ws typed for one group (Afric, non-afric), esti- mtes of vribility (see below) were clculted for ech popultion seprtely nd subsequently verged. This tretment ws chosen to void bised vribility estimte due to popultion substructure (Whlund effect). Furthermore, this strtegy ccounted for the fct tht n unequl number of popultions were typed for different loci. Vribility mesures, recombintion rte, nd corrections for effective popultion sizes of X chromosomes nd uto- somes: Two mesures of microstellite vribility were used in this study: vrince in repet number (Goldstein et l. 1995) nd heterozygosity. Both mesures were corrected for smll smple sizes by multiplying by n/(n 1), where n is the number of typed chromosomes. To ccount for the drmtic influence of the repet number on muttion rtes (Goldstein nd Clrk 1995; Hrr nd Schlötterer 2000; Schlötterer 2000), we normlized the vrince in repet number for ech locus by dividing by the verge number of repets (V LC ). This correction ccounts for substntil prt of the heterogeneity in microstellite vribility mong loci (Schlötterer 2000). Throughout this text we use V LC s synonym for vrince in repet number. Furthermore, only microstellites with 15 repets were used in this study becuse longer microstellites tend to be exponentilly more vrible (Brinkmnn et l. 1998). Vrince in repet number, heterozygosity, nd recombintion rte (djusted for no recombintion in mles) for ll loci re listed s supplementl tble t the Genetics website t To ccount for different effective popultion sizes of X chromosomes nd utosomes we introduced correction fctor for the X chromosoml vribility mesures. The correction fctor ws equl to the rtio of the effective popultion sizes of utosomes to X chromosomes. This rtio is RESULTS Levels of vribility in Africn nd non-africn popultions: We nlyzed 133 microstellite loci, of which 40 loci were locted on the X chromosome nd 93 on the utosomes. A complete list of loci nd their vribility in Africn nd non-africn popultions is vilble s supplement t The joint nlysis of ll microstellite loci indicted tht both mesurements of vribility, heterozygosity, nd vrince in repet number (corrected for the repet number V LC ) were significntly higher in Africn pop- ultions thn in non-africn ones. This difference between Africn nd non-africn popultions ws still sig- nificnt if utosoml nd X chromosoml microstellite loci were nlyzed seprtely (P 0.001, Mnn Whitney U-test, Tble 2). This pttern is consistent with recent comprisons of Africn nd non-africn popultions, which mostly detected higher levels of vribility in Afri- cn popultions (Begun nd Aqudro 1993, 1995b; Agudé 1998, 1999). X chromosoml vribility is more reduced thn u- tosoml in non-africn popultions: While both X chro- mosoml nd utosoml loci hd significnt reduction in vribility in non-africn popultions, closer inspeck N A 8(N ef 2N em) N X 9(N ef N em ), (1) tion of the dt indicted tht the reltive reduction in vribility differed between X chromosomes nd utowhere N ef nd N em re the effective popultion sizes of femles nd mles, respectively (Cbllero 1994). Assuming blnced sex rtio, the correction fctor k ws The djusted estimtes of vribility were clculted s nd 1 H corr 1 1 k(1/(1 H obs ) 2 1) (2) V corr kv obs. (3) somes (Figure 1, Tble 2). X chromosoml microstellites re more vrible thn utosomes in Africn popu- ltions, but in non-africn popultions X chromosomes re slightly less polymorphic thn utosomes. Despite the opposite trend in the two popultions, heterozygosities re significntly different between X chromosomes nd utosomes in both Africn nd non-africn popul- tions (P 0.005, Mnn Whitney U-test). V LC showed, especilly in Africn popultions, similr trend, but the

4 250 M. Kuer et l. TABLE 2 Differences of utosoml nd X chromosoml microstellite vribility in Africn nd non-africn popultions Men heterozygosity Men V LC Popultion Autosome X chromosome Difference A:X Autosome X chromosome Difference A:X Afric 0.62 (0.23) 0.75 (0.12) ** 0.67 (0.86) 0.84 (0.84) P 0.07 Non-Afric 0.52 (0.22) 0.4 (0.2) ** 0.32 (0.45) 0.24 (0.46) NS Difference Afric-non-Afric ** ** ** ** Vribilities re not corrected for effective popultion sizes of the chromosomes. Vlues in prentheses re stndrd devitions of the men. ** P 0.01 (Mnn Whitney U-test). A:X, utosomes:x chromosome; NS, not significnt. V LC, length corrected vrince in repet number. difference ws not sttisticlly significnt. An implic- bintion rte is predicted we tested for such correltion tion from these results is tht vribility of microstellites on utosomes nd X chromosomes in Africn nd locted on the X chromosome is more strongly non-africn popultions. In Africn popultions we observed reduced in non-africn popultions. To test for sttisticl n lmost significnt correltion only between significnce we performed n ANOVA nd found one mesure of vribility (V LC ) nd recombintion rte significnt interction between the fctors chromosome for X chromosoml loci (r 0.31, P 0.056; Spermn (X utosomes) nd popultion (Afric non-afric) rnk correltion). Interestingly, this correltion ws on vribility (H, P ; V LC, P 0.05; Tble 3). more pronounced for the non-africn X chromosoml Up to now, vribility mesures were not corrected microstellites (H, r 0.49, P 0.05; V LC, r 0.43, for the different effective popultion sizes of X chromosomes P 0.05; Spermn rnk correltion). No significnt nd utosomes. Assuming blnced sex rtio correltion for vribility (H nd V LC ) nd recombin- (n equl number of mles nd femles), the effective tion could be detected for either Africn or non-africn popultion size of utosomes is 1.33 times lrger thn utosomes (non-afric r 0.04, P 0.69; Afric r the effective popultion size of X chromosomes. After 0.01, P 0.95; Spermn rnk correltion). ccounting for these differences in effective popultion Different effective popultion sizes of chromosomes: size (see mterils nd methods), X chromosoml The reltive level of vribility on X chromosomes nd nd utosoml vribility ws similr in non-africn pop- utosomes is strongly ffected by the sex rtio, which ultions (Tble 4). The difference between the chromosomes in turn depends on the reproductive success of ech in the Africn popultions, however, becme sex. Therefore, we tested whether different effective more significnt. popultion sizes of mles nd femles could explin our Recombintion rte nd vribility: An importnt s- results. Our tests re bsed on correction fctor for pect of our experimentl design ws to include loci from the different effective popultion sizes of the sex chro- wide rnge of recombintion rtes, but lso to select mosomes under vrious sex rtios. chromosoml regions with comprble recombintion In the simple cse of blnced sex rtio, the excess rtes (fter correction for bsence of recombintion in of vribility on Africn X chromosomes is sttisticlly mles) on both chromosomes. Since under both selection significnt (Tble 4). In non-africn popultions we models correltion between vribility nd recom- noted reduced heterozygosity of X-linked microstel- Figure 1. Microstellite vribility on the X chromosome (blck brs) nd utosomes (gry brs) in Africn nd non-africn popultions. The error brs indicte the 95% confidence intervl of the men heterozygosity (not corrected for different effective popultion sizes of X chromosomes nd utosomes). The difference between the X chromosome nd utosomes in both Africn nd non-africn popultions is sttisticlly significnt (P 0.005, Mnn Whitney U-test).

5 Microstellite Vribility in D. melnogster 251 TABLE 3 ANOVA for the impct of chromosome nd popultion on vribility Heterozygosity V LC b Fctor d.f. Men squre F P d.f. Men squre F P Chromosome Popultion Chromosome popultion Heterozygosity ws rcsine trnsformed. b V LC (length corrected vrince in repet number) ws log trnsformed. cently obtined from survey of published D. melnogster sequences (Andolftto 2001). The sequence nly- sis of 40 genes in non-africn popultions of D. simulns, close reltive of D. melnogster, which lso recently spred from Afric, lso reveled lower levels of vribility on the X chromosome (Begun nd Whitley 2000). This consistency of vrious dt sets strongly suggests common underlying biologicl process. In the following we discuss processes tht could, in principle, explin the observed distribution of vribility. Neutrl explntions (demogrphic effects): Sex rtio: Mle D. melnogster hve one X chromosome, while femles crry two X chromosomes. Thus, the effective popultion size of X chromosomes is dependent on the vrince of reproductive success of mles nd femles. For Africn (Wu et l. 1995; Cpy et l. 2000) nd non- Africn (Korol et l. 2000) popultions, mte selection hs been reported. Furthermore, it hs been proposed tht due to mte selection X chromosomes cn hrbor 90% (rther thn 75%) of the utosoml vribility (Nunney 1993; Chrlesworth 2001). At lest in Europen D. melnogster popultions, the effect could be countered by lrge vrition in reproductive success mong femles (Bouletreu 1978). In summry, there is substntil evidence for vrince in reproductive success in D. melnogster, but the extent to which differentil reproductive success skews the rtio of X chromo- soml to utosoml vribility is simply not known. Our dt indicte tht, in non-africn popultions, levels of vribility were no longer significntly different mong chromosomes when blnced sex rtio is s- lites. This difference, however, ws not sttisticlly significnt. Given tht the rtio of the X chromosome-utosome vribility is converse in Africn nd non-africn popultions we must ssume inverted sex rtios in these popultions. Africn popultions should hve n excess of femles while in non-africn popultions the effective popultion size of mles should be lrger. Becuse we did not detect significnt difference between utosoml nd X chromosoml vribility in non-africn popultions, we did not further pursue the possibility of lrger effective popultion size of mles in non-africn popultions. To explore the possibility of nonblnced sex rtio in Africn popultions, we ssumed lrger effective popultion size in femles. Both vribility estimtors, heterozygosity nd V LC, were still significntly higher on Africn X chromosomes when we ssumed five times lrger effective popultion size of femles (Tble 4). However, even n ssumed 50-fold higher femle popultion size could not ccount for the observed heterozygosities. DISCUSSION Our nlysis of lrge number of microstellite loci in Africn nd non-africn D. melnogster popultions indicted tht the level of vribility differed between X chromosomes nd utosomes. While X chromosomes were significntly more vrible in Africn popultions, non-africn popultions hd higher heterozygosities for utosoml microstellite loci. Similr results were re- TABLE 4 Microstellite vribility in Africn nd non-africn popultions fter correction for different effective popultion sizes of utosomes nd X chromosomes Men heterozygosity Men V LC Popultion Sex rtio ( f:m) Autosome X chromosome Difference A:X Autosome X chromosome Difference A:X Afric 1: (0.23) 0.78 (0.11) ** 0.67 (0.86) 1.12 (1.1) ** Non-Afric 1: (0.22) 0.44 (0.20) P (0.45) 0.32 (0.62) NS Afric 5: (0.23) 0.75 (0.12) ** 0.67 (0.86) 0.85 (0.85) * ** P 0.01, * P 0.05 (Mnn Whitney U-test). Vlues in prentheses re stndrd devitions of the men. f:m, femles:mles; A:X, utosomes:x chromosomes; NS, not significnt. V LC, length corrected vrince in repet number.

6 252 M. Kuer et l. correltion between vribility nd recombintion rte. The correltion between recombintion nd levels of vribility hs been explined by selective sweeps of beneficil muttions (Begun nd Aqudro 1992) or bck- ground selection (Chrlesworth et l. 1993). Our observtion tht significnt correltion exists on the X chromosome suggests tht selection hs greter effect on the non-africn X chromosome thn on utosomes, but provides no further insight whether the fixtion of beneficil or the removl of deleterious muttions is determining the pttern of vribility. Differentil selective forces re operting in Africn nd non-africn popultions: Any study ttempting to explin the impct of selection on utosoml nd X chromosoml genes hs to consider the effect of dominnce. While the dynmics of dominnt muttions will not differ substntilly mong X chromosomes nd utosomes, recessive muttions will be selected more effi- ciently when locted on the X chromosome. Recently, Begun nd Whitley (2000) reviewed the consequences of dominnce effects for levels of vribility under vrious selection regimes. For the reminder of the text, however, we ssume tht muttions re recessive. Evidence for multiple beneficil muttions on non-africn X chromosomes: The genetic consequences of exposure to novel hbitt re well investigted in simple orgnisms, such s Escherichi coli nd yest. Hbitt shifts were shown to increse the fixtion rte of beneficil mut- tions, leding to higher fitness in the new environment (Novick nd Szilrd 1950; Lenski nd Trvisno 1994; Imhof nd Schlötterer 2001). Similrly, the coloniztion of more temperte environments outside of Afric hs undoubtedly resulted in n incresed potentil for the fixtion of beneficil muttions in D. melnogster. In principle, two different scenrios hve to be distinguished. First, the beneficil muttions re novel nd occurred during the coloniztion event. Second, the beneficil muttions were lredy present in the founder popultions, but only with the hbitt shift did these muttions become dvntgeous. While, for the first scenrio, lower X-linked vribility could be ssumed (Chrlesworth et l. 1987), the predictions re less cler for the second model. Orr nd Betncourt (2001) considered sudden environmentl chnge, which rendered previously deleterious lleles (in muttion-selection equilibrium) beneficil (Orr nd Betncourt 2001). Under this model, the uthors showed tht stronger reduction in vribility on the X chromo- some my or my not be expected if dptive muttions involve previously deleterious lleles. Until now, literlly nothing hs been known bout muttions tht re beneficil in novel environment nd the chrcteriztion of those muttions is mong the most exciting chllenges to evolutionry biologists. Despite lcking cler prediction on the nture of beneficil muttions, we tested whether the pttern of vri- bility is consistent with beneficil muttions reducing sumed. For Africn X chromosomes we found tht neither blnced sex rtio nor fivefold excess of femles could explin the higher vribility on Africn X chromosomes. On the bsis of the vilble dt, it is not possible to decide whether the sex rtio in Africn D. melnogster popultions is even more bised thn we ssumed. Nevertheless, fivefold excess of femles is more conservtive thn in previous reports, which suggested tht X chromosoml vribility should be 90% of the utosoml vribility (Nunney 1993; Chrlesworth 2001), which corresponds to three fold reduction in mle effective popultion size. Founder effect: It is ssumed tht D. melnogster strted from Afric to colonize the rest of the world 10,000 yers go (Dvid nd Cpy 1988). Such founder effects re often ssocited with loss of genetic vribility. Becuse our dt lso indicted loss of vribility in the non-africn popultions, we were interested whether bottleneck could explin the loss of vribility. We pursued two different pproches to test for bottleneck in the non-africn popultions. Our first test tkes dvntge of the well-investigted reltionship between the number of lleles nd heterozygosity. After bottleneck, heterozygosity excess is expected t neutrl loci (Cornuet nd Luikrt 1996). We used the three different test sttistics, sign test, stndrdized difference test, nd Wilcoxon test, ll implemented in the Bottleneck 1.2 softwre (Cornuet nd Luikrt 1996). Irrespective of the muttion model used (strict stepwise or two-phse model with 30% nonstepwise muttions) we did not detect significnt heterozygosity excess in the non-africn popultions (P 0.05). The second test ws bsed on the property of bottlenecks tht they re genome-wide phenomenon. Hence, if founder event cused the reduction in vribility, then ll microstellite loci locted on the sme chromosome should be ffected to the sme extent. To test this, we plotted the observed heterozygosity in non-africn nd Africn popultions ginst recombintion rte. If the reduction in vribility were entirely due to bottleneck, no correltion between vribility nd recombintion rte should be detected. Interestingly, we observed significnt correltion between recombintion rte nd heterozygosity on the X chromosome only in non-africn popultions (non-afric r 0.49, P 0.001; Afric r 0.08, P 0.64; Spermn rnk correltion; Figure 2). The sme effect cn lso be seen for V LC in non-africn popultions (r 0.43, P 0.005; Spermn rnk correltion), where, in contrst, mrginlly significnt correltion is lso observed in Africn popultions (r 0.31, P 0.056; Spermn rnk correltion). On the utosomes, no correltion could be detected (non-afric r 0.04, P 0.69; Afric r 0.01, P 0.95; Spermn rnk correltion; Figure 2b). This test shows tht simple chnges of popultion size cnnot explin our dt. Furthermore, even reduction in the effective femle popultion size would not hve produced

7 Microstellite Vribility in D. melnogster 253 Figure 2. Heterozygosity of non-africn nd Africn popultions plotted ginst recombintion rte [recombintion events/(kilobse genertion)]. Crosses nd the dshed line represent Africn, nd dots nd the solid line non-africn, popultions. To ccount for the bsence of recombintion in mles, we corrected the recombintion rtes of utosomes nd X chromosomes, ssuming 1:1 sex rtio. (A) A significnt correltion between heterozygosity nd recombintion rte ws observed for non-africn X chromosoml loci (non-afric r 0.49, P 0.001; Afric r 0.08, P 0.64; Spermn rnk correltion). (B) On the utosomes no correltion could be detected (non- Afric r 0.04, P 0.69; Afric r 0.01, P 0.95; Spermn rnk correltion). flects the vrition of RV LC vlues on X chromosomes nd utosomes. Consistent with higher impct of selective sweeps on the X chromosome, we found lrger vrince of RV LC vlues on the X chromosome. Nevertheless, this result does not indicte whether the beneficil muttions occurring on the X chromosome were lredy segregting in the Africn popultion or re of recent origin. However, it should be noted tht our dt do not suggest tht lrger number of beneficil muttions occurred on the X chromosome thn on utosomes. Due only to the lrger hitchhiking effect of beneficil muttions on the X chromosome is their presence more obvi- ous. We ssume tht similr number of beneficil muttions (per gene) lso occurred on the utosomes, but much smller genomic region hs hitchhiked with them. The pttern of vribility in Africn popultions: One further importnt result of our survey is tht in Africn popultions X chromosoml microstellites hve sig- nificntly higher vribility thn utosomes. Recently, Andolftto (2001), who obtined similr pttern in survey of published sequence dt, proposed tht utosoml vribility in Africn popultions could be X chromosoml vrition in non-africn popultions. Becuse microstellite loci re neutrl mrkers, reduced microstellite vribility t given locus is most likely cused by linkge to selected site. Thus, the prediction is tht some microstellites on the X chromosome will be linked to beneficil muttion, while others re not linked. Given tht mny beneficil muttions re required to reduce the vribility on the X chromosome, we expect reduced vribility t severl microstellite loci. Consequently, microstellite vribility should show lrge vrition mong loci. Becuse utosomes did not experience strong reduction in vribility, fewer microstellite loci re expected to be ssocited with beneficil muttion. Hence, the vrition in vribility mong loci is expected to be less pronounced mong utosoml loci thn mong X chromosoml loci. To test this we clculted the vrince of RV LC (RV LC V na /V Afr ) vlues for X chromosoml nd utosoml loci. To obtin confidence intervl for this vrince we generted 100 pseudoreplics by bootstrpping the RV vlues for ech group of loci. Figure 3 shows the men of the vrince of RV LC nd its 95% confidence intervl. This vlue re-

8 254 M. Kuer et l. Figure 3. Men vrince of 100 resmples of RV LC (RV LC V LCnA /V LCAfr ) vlues for X chromosomes nd utosomes. The error brs indicte the 95% confidence intervl of the men RV LC. for which clines with high frequencies in Africn popultions were described. Becuse most of the lines used were not kept live, we did not determine the inversion sttus of ech line. Thus, the inversion frequencies in our Africn smples re not known. However, if these inversions hve ffected our vribility estimtes, this should be recognizble even without the knowledge of the inversion sttus of ech of the lines nlyzed. We split the utosoml microstellites into loci, which re locted outside nd within genomic regions hrboring these inversions. No loci were found to mp to the inversion brekpoints. Our microstellite set contined only three of the five inversion loctions known to rech high frequencies in Afric. The chromosoml loctions of the inversions, s well s the microstellite vribilities within nd outside these regions, re given in Tble 5. In contrst to the expecttions for recently swept inversions, we did not detect ny evidence for reduction in vribility of microstellites locted within the chro- mosoml regions to be possibly n inversion. This observtion holds irrespective of whether we nlyzed microstellites within ech inversion individully or ll three inversions jointly nd compred them to ll microstel- depressed by recent increse in frequency of chromosoml inversions in these popultions. In fct severl lites locted outside of the inversion regions (Tble 5). inversions hve been found to form clines with higher While the seprte nlysis of microstellite loci frequencies t low ltitudes ner the equtor (Lemeu- within inversions nd outside inversions seems not to nier nd Aulrd 1992). Nvrro et l. (2000) found support the hypothesis of Andolftto (2001), we note tht vribility levels within inversions could be reduced tht our test is rther crude nd restricted to the impct s long s the inversion hs not reched equilibrium of common cosmopolitn inversions. However, n lter- (Nvrro et l. 2000). Experimentl dt, however, in- ntive explntion of the dt my be tht the pttern dicte tht reduction of vribility is confined to the of vribility in Africn popultions is consistent with proximity of the inversion brekpoints. No reduced vri- bckground selection. According to the bckground sebility ws detected in the middle of inversions (Hsson lection model, X chromosomes should hrbor more nd Enes 1996; Agudé 1998, 1999; Andolftto et neutrl vrition thn utosomes (Chrlesworth et l. l. 1999; Bénssi et l. 1999; Depulis et l. 1999). 1987). Recessive deleterious lleles re purged more Furthermore, n interesting finding of Andolftto efficiently from X chromosomes nd, thus, fewer neutrl (2001) ws tht D. simulns, sibling species of D. melno- lleles re removed together with the deleterious lleles. gster, which shows very few inversion polymorphisms, showed no elevted X-linked vribility in Africn popu- ltions. Given the possible implictions of the dt, we tested for n influence of common cosmopolitn inversions, CONCLUSION Our results re consistent with the interprettion tht, in the Africn popultions, which re presumbly close TABLE 5 Microstellite vribilities within nd outside of known inversion loctions in Africn popultions Subsmple Cytologicl loction No. of microstellites Men H Men V LC In(2L)t 22D 34A (0.24) 0.98 (1.18) In(2R)NS 52A 56F (0.28) 0.33 (0.28) In(3L)P 63C 72E (0.06) 0.61 (0.39) All inversions (0.24) 0.71 (0.91) No inversions (0.23) 0.64 (0.82) All loci (0.23) 0.67 (0.86) Pirwise differences of men H nd V LC between ll subsmples were not significnt (Mnn Whitney U-test, P 0.1). Vlues in prentheses re stndrd devitions of the men. V LC, length corrected vrince in repet number.

9 Microstellite Vribility in D. melnogster 255 to n equilibrium sitution, bckground selection is the Brvermn, J. M., R. R. Hudson, N. L. Kpln, C. H. Lngley nd W. Stephn, 1995 The hitchhiking effect on the sites frequency evolutionry force shping the prtitioning of vribility spectrum of DNA polymorphisms. Genetics 140: mong X chromosomes nd utosomes. Non-Africn Brinkmnn, B., M. Klintschr, F. Neuhuber, J. Huhne nd B. Rolf, popultions, in contrst, seem to be more ffected by 1998 Muttion rte in humn microstellites: influence of the structure nd length of the tndem repet. Am. J. Hum. Genet. the spred of beneficil muttions tht were most likely 62: ssocited with the hbitt expnsion of D. melnogster. Cbllero, A., 1994 Developments in the prediction of effective However, the influence of evolutionry forces, such s popultion size. Heredity 73: Cpy, P., M. Veuille, M. Pillette, J. M. Jllon, J. Vouidibio et vrition in reproductive success, chromosoml inver- l., 2000 Sexul isoltion of geneticlly differentited symptric sions, nd founder effects on the observed pttern of popultions of Drosophil melnogster in Brzzville, Congo: the vribility, is not cler. More theoreticl nlyses re first step towrds specition? Heredity 84: Chrlesworth, B., 2001 The effect of life-history nd mode of inherirequired to model the expecttions for X chromosoml tnce on neutrl genetic vribility. Genet. Res. 77: nd utosoml vrition in equilibrium nd nonequilibtive Chrlesworth, B., J. A. Coyne nd N. H. Brton, 1987 The rel- rtes of evolution of sex chromosomes nd utosomes. Am. rium popultions. Nt. 130: We re grteful to T. Hrr, R. Bürger, nd the members of the C.S. Chrlesworth, B., M. T. Morgn nd D. Chrlesworth, 1993 lb for criticl discussions t vrious stges of the project. T. Wiehe, The effect of deleterious muttions on neutrl moleculr vri- B. Chrlesworth, nd nonymous reviewers provided helpful com- tion. Genetics 134: ments on the mnuscript. This work hs been supported through Comeron, J. M., M. Kreitmn nd M. Agude, 1999 Nturl selec- Fonds zur Förderung der wissenschftlichen Forschung grnts to C.S. tion on synonymous sites is correlted with gene length nd recombintion in Drosophil. Genetics 151: Cornuet, J. M., nd G. Luikrt, 1996 Description nd power nlysis of two tests for detecting recent popultion bottlenecks from LITERATURE CITED llele frequency dt. Genetics 144: Crow, J. F., nd M. Kimur, 1970 An Introduction to Popultion Genetics Agudé,M.,1998 Different forces drive the evolution of the Acp26A Theory. Hrper & Row, New York. nd Acp26Ab ccessory glnd genes in the Drosophil melnogster Dvid, J. R., nd P. Cpy, 1988 Genetic vrition of Drosophil melnogster species complex. Genetics 150: nturl popultions. Trends Genet. 4: Agudé, M., 1999 Positive selection drives the evolution of the Acp- Depulis, F., L. Brzier nd M. 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