The Evolution of Reproductive Isolation as a Correlated Character Under Sympatric Conditions: Experimental Evidence Author(s): William R. Rice and George W. Salt Source: Evolution, Vol. 44, No. 5 (Aug., 1990), pp. 1140-1152 Published by: Society for the Study of Evolution Stable URL: http://www.jstor.org/stable/2409278 Accessed: 25-03-2015 19:26 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. Society for the Study of Evolution is collaborating with JSTOR to digitize, preserve and extend access to Evolution. http://www.jstor.org This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions Evolution,44(5), 1990, pp. 1140-1152 THE EVOLUTION OF REPRODUCTIVE ISOLATION AS A CORRELATED CHARACTER UNDER SYMPATRIC CONDITIONS: EXPERIMENTAL EVIDENCE WILLIAM R. RICE' of New Mexico, Albuquerque,NM 87131 USA 'Departmentof Biology, University AND GEORGE W. SALT of California,Davis, CA 95616 USA 2Departmentof Zoology, University Abstract.-A set of experimentsis describedthat teststhe generalhypothesisthat sympatricspeciation is geneticallyfeasible wheneverreproductiveisolation evolves indirectlyas a correlated character.We specificallytest the hypothesisthat disruptiveselection on habitat preferencecan lead to sympatricspeciationwhenindividualsmate locallywithintheirselectedhabitat.Drosophila melanogasterwas used as a model system.A 35-generationexperimentusing a complex habitat spatiotemporal maze led to completereproductiveisolationbetweensubpopulationsusingdifferent habitats.The reproductiveisolation thatdeveloped over the course of the experimentwas a result to mate in thehabitattypeselectedbytheirparents,i.e., a gradualbreakdown of offspring returning in migrationbetweenhabitats. Received May 1, 1989. Accepted December 7, 1989. Surprisinglittle is known about the genetic processes leading to speciation. One of the major controversiesconcerningspeciation is whetheror not geographicalseparation (or more generallyallopatry) is a prerequisiteto the speciationprocess.Mayr (1942, 1947, 1963, 1970, 1982) arguedthat withthe exceptionof instantaneousspeciation via polyploidy,all observed cases of speciationare consistentwiththethesisthat a physical barrierhad firstpreventedgene flow between subunits of a species before reproductive isolation evolved between them. Mayr does not precludethe possibilityof sympatricspeciation,but arguesthat available experimentaland observational evidence does not support its having occurred.Because Mayr's assertionof the virtual universalityofallopatricspeciationhas been widely accepted (e.g., Futuyma and Mayer, 1980; Paterson, 1981; Templeton, 1981; Carson, 1987), we will referto it as the "allopatryparadigm." The allopatry paradigm has been both challengedand supportedby empiricaland theoreticalstudies(forreviewssee Futuyma and Mayer, 1980; Templeton, 1981; Bush and Howard, 1986). More recenttheoretical ' Present address: Biology Board of Studies, Universityof California,Santa Cruz, CA 95064 USA. work by Slatkin (1982) and Rice (1984, 1987), however, indicates that sympatric speciation should be biologically feasible when reproductiveisolation evolves indirectlyas a correlatedcharacter.The foundation for this conclusion is that the evolution of reproductive isolation as a correlatedcharactereliminates an antagonisticinteractionbetweenselectionand recombination [the selection-recombination antagonism (Felsenstein, 1981)] that appears to be the principalgeneticobstacle to sympatricspeciation. Here we describea long-termexperimental studythatteststhe hypothesisthat speciation can resultfromdisruptiveselection on habitatpreferencewhen organismsmate locally aftertheyhave assorted themselves into the environment.In this case reproductive isolation evolves indirectlyas a byproductof habitatspecialization.That is, a barrierto gene flow is graduallyproduced as offspringincreasinglyreturn to mate withinthe habitattypethatwas selectedby theirparents. A preliminaryexperimentsupportedthe hypothesis (Rice, 1985), but this experiment had importantlimitations.These include (1) a supergenemarkersystem,used to measure gene flow,that preventedthe exchange of X chromosomes between the habitats, subpopulations utilizingdifferent 1140 This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions EVOLUTION OF REPRODUCTIVE (2) changes in the experimentalprotocol duringthecourse oftheexperiment,and (3) the fact that only very strong selection against "habitat switching"(i.e., selection originatingas eggs in one against offspring habitatbut choosinganotherhabitatforreproduction)was studied. All of these problems are eliminatedin the experimentsrereport,describing portedhere.A preliminary some aspects of the earlygenerationsof the theseexperiments,can be foundin Rice and Salt (1988). To avoid confusionwe explicitlydefine severalterms.Ratherthanprovide our own idiosyncraticdefinitions,we use those of Bush and Howard (1986). allopatric: "Allopatric populations are separatedby uninhabitatedspace (even ifit is a very shortdistance), across which migration (movement) occurs at a very low frequency." sympatric:"two populations are sympatric if individuals of each are physicallycapable of encounteringone another... with moderatelyhigh frequency." species: "a group of populations whose evolutionarypathwayis distinctand independentfromthatofothergroups;a distinct and independentevolutionarypathway is achieved by the group's reproductiveisolation fromother groups.... groups have acheived full species status if they are or could be (giventheopportunity)truelysympatric withoutlosing theirseparate identities throughinterbreeding." NATURAL HISTORY CONTEXT The experimentswere designed to simulate the followingnaturalhistorycontext. Consider a large nascent island that is isolated and that has recentlybeen colonized by many tree species. Because of its young age, the island is presumed to have a depauperate frugivorefauna and therefore contains many "empty niches." Next suppose that a frugivorousflypopulation colonizes the island. The colonizing flypopulation is assumed to be adapted to fruit species thatare not foundon theisland,but to be sufficiently preadapted to (1) be capable of utilizing two of the extant tree species (presumedto be rareand widelydispersedthroughouttheisland),whichwe will referto as whiteand black fruit,and (2) have ISOLATION 1141 sufficientpolygenic variabilityfor habitat preferenceso thatat least some individuals of the founderflypopulation locate white, and othersblack fruit. Lastly, we assume that the founder fly populationmateslocallyon itsselectedfruit resource,and thatthe whiteand black fruit spatiotemporalhabare located in different itats,so that the behavioral and phenological phenotypes required to locate white (black) fruitare positive (negative) phototaxis, negative (positive) geotaxis, white (black fruit-specific)chemofruit-specific taxis,and eclosion duringthelatter(earlier) part of the annual cycle. The rationale for theseformsofselectioncan be foundin Rice (1984, 1985, 1987), and a moregeneralnatural historycontextcan be found in Bush (1969), Tauber and Tauber (1977), Moore (1979), Rausher (1984), and Jaenike(1985, 1986). The simultaneouspresence of both suitable fruit resources generates disruptive selectionon spatiotemporalhabitat preference. Flies selecting intermediatespatiotemporalhabitatswill findneithersuitable perish.Whenthe resourceand willtherefore fliesmate locally on theirselectedresource, as commonly occurs in frugivorousflies (Bush, 1969), therewill be positive assortativematingbetweenindividualswithsimilar habitat preference.In this context resource specialization can graduallylead to reproductiveisolation via the diminished increasmigrationthataccrues as offspring inglyreturnto, and mate within,thehabitat type selected by theirparents. The laboratoryexperimentsdescribedbelow attemptto mimictheabove naturalhistory context. We specificallyask whether thefounderflypopulationwill splitintotwo reproductivelyisolated resourcespecialists. In this context reproductive isolation evolves as a correlatedresponse to habitat specialization. MATERIALS ANDMETHODS General CultureMethods The common fruitfly(Drosophila melanogaster)was used as a model system.The stockused to begintheexperimentswas derived from30 mated femalescollectednear Davis, Californiain 1984. This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions 1142 W. R. RICE AND G. W. SALT Three groupsof 120 mated femaleswere randomly selected from this stock. They were placed for24 hr,fivefemalesper vial, into 8-dram vials containing 12 ml of instant Drosophila food (Carolina Biological Supply Inc.). A tygontube,withoutside diametermatchingthe inside diameterof the 8-dram culturevial, was insertedto a positionjust above the level of the food. This provideda removableinteriorwall. The larvae developed in the food (storedin a 25?C incubator) and, when mature, crawled up onto the tygon tube and attached themselves as pupae. The tygontubes containing the pupae were removed fromthe vials just beforeeclosion and thenplaced into one of threepopulation mazes (all of the same design) that simulated the island naturalhistorycontext. The Population Mazes A detailed drawingof a population maze can be foundin Rice and Salt (1988). Each maze was located in the same controlled environmentalroom witha temperatureof 25?C and a relativehumidityof 50%. Lighting was continuousfromceilingfluorescent lights.Betweengenerationseach maze was completelydisassembled and washed with warm tap water. To begin everygenerationof the experiment,each of the threesets of tygontubes is placed in the centerof its maze and attached to a manifoldthatleads to a central chamber.This chambergradesfroma lighted to a darkenedend, and separatestheflies based on their phototacticbehavior. The fliesare next separatedbased on theirgeotactic behavior by the uprightsections betweenthefourbulbous quadrantsofa maze. Resolution of fliesbased on chemotaxis is next accomplished by two ports separated by a vent. One port emanates the odor of ethanol and the otheracetaldehyde.These odorantsare provided via a wick thatoriginates in a small glass canister that is attached to each food vial. The food vials are referredto as "habitats," and are labeled one througheight.Lastly,the fliesare separated based on theirdevelopmenttime by collectingfliesfromthehabitatsthreetimes [early(E: the tenthday of the 14-day generationcycle), middle (M: days 1-13), and late (L: day 14)]. To preventovercrowding oftheflies,themiddlecollectionwas further divided into five sequential collections. In summary,the mazes separatedthefliesinto 24 spatiotemporal habitats [(eight spatial habitats) x (threetemporalcollections)]. There were 12 one-way funneltraps located withineach maze. One was placed at the entryportto each quadrant,and one at the top of each collection vial (habitat). These wereused to preventfliesfromchoosing more than one habitat in a singlegeneration.The rationale forthis constraintis that the mazes only contained one habitat of each type,and thereforemovement between habitats of the same type was precluded. Selection To produce disruptiveselectionon habitatpreference,60 mated femaleseach from habitats5-early(SE, simulatingblack fruit) and 4-late (4L, simulatingwhitefruit)were used to propagatethe nextgeneration.Flies selectingother habitats were presumed to have perishedsince theydid not finda suitable resource.To stimulatetheindependent carryingcapacities that the spatially and temporally separated resources would be expected to produce,the 60 mated females fromhabitats SE were culturedseparately fromthose from4L. Sometimes, especially during the early generationsof the experiment,fewerthan 60 mated femaleswerefoundin habitatsSE and 4L. In these instances additional flies were taken from a neighboringhabitat to ensure that a total of 120 mated females were used to produce each generation.For exampleiffewerthan60 femaleswerefound in habitat SE, then we took additional females fromhabitat 6E; and if there were too few additional fliesin 6E then we also took fliesfromthe earliestof the sequential collectionsofhabitatSM. This poolingprocedureconservatively weakenedthestrength ofselectionbut suppressedthedevelopment of inbreedingdepressionduringthe course of this long-termexperiment. A second complicationin the experimental protocolwas thatover the course of the experiment development time was occasionallyunusuallyfastor slow. This almost This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions EVOLUTION OF REPRODUCTIVE certainlyoccurreddue to minorfluctuations in temperaturecontroloftheincubatorand/ or thetemperature-controlled room. We adjusted for this by pooling the E collection withthe firstofthe sequentialM collections when the flies were developmentally advanced and by poolingtheL collectionwith thelast ofthesequentialMcollections when thefliesweredevelopmentallydelayed. The rationale for this adjustment is that temperaturevariation in nature would be expected to affectthe phenology simultaneously of both the flies and their fruit resources. A second formof selectionthatmightbe expectedto occur,in addition to disruptive selectionon habitatpreference,is selection against "habitat switching."If the habitats differin theirabiotic and/orbiotic conditions,then each habitatwould be expected to have an idiosyncraticselection regime. For example, suppose the two fruitswere protectedbydifferent toxiccompounds that required differentcoadapted gene complexes to be effectively detoxified.A flyoriginatingas a larva on white fruitwould be expected, on average, to be more adapted to utilize this fruitthan a flyoriginatingon black fruit,and vice versa. A flythat returnedto the resourcetype selected by its to have parentswould be expectedtherefore higherfitnessthanone switchingto the other fruittype.Alternatively, selectionagainst habitat switchingwould be nominal when the environmental conditions associated withthetwo suitablehabitatswerevirtually identical. To exploretheimpactof selectionagainst habitatswitching,in one experimentwe applied strong selection against habitat switchingand in the other we applied no such penalty. To produce strongselection against habitat switchingwe removed all femalesthatselecteda habitatdifferent from the one chosen by her parents.Males were not penalized. This produced an average 50% reductionin fitnessforhabitatswitching. In a thirdexperiment,the control,there was no disruptiveselectionon habitatpreference and no selection against habitat switching.In thecontrolall of thefliesfrom all the habitatswere mixed in a singlecon- ISOLATION 1143 tainerand 120 matedfemaleswererandomlyselectedto propagateeach successivegeneration. Sixtymated femaleswere cultured on to standardmedium and the other60 on to kynurenine-supplemented medium (see below). Because ofbudgetaryconstraints, thelevel of technical supportneeded to be reduced startingin generation26 of the experiment. This was accomplished by not passing the controlpopulationthroughitshabitatmaze between generations26 and 33. The protocol for handling the control population was not changed in any other way. This changeis expectedto have had virtuallyno effecton the controlpopulation, however, since habitat choice by these flieswas obviated by randomlyselectingflies for culturingeach generation.As a consequence of this change, the graphs depicting habitat preferenceof the controlpopulation do not have data forgenerations26-33. In summary,threelevels ofselectionwere applied in threeparallel experiments.The controlhad no artificialselection. One experimental population (the double-selection experiment)had selection applied to both habitatpreferenceand habitatswitching, and the other (the single-selectionexperiment) only had selection on habitat preference. Measuring Philopatry Reproductive isolation was expected to evolve in the experimentalsystemvia philopatry,i.e., offspring returningto mate in the habitat type selected by theirparents. Because fliesmated locally withintheirselected habitats (see below), habitat preference necessarilyled to positive assortative matingbetween individuals with the same habitat preferencephenotype. A phenocopytechniquewas used to measure philopatry.We firstrecombined the mutationsvermilion(v, I-33.0 blockingthe productionof the brown-eyepigment)and raspberry(ras, I-32.8, blockingthe productionofthered-eyepigment)intothefounder stock. In all crosses the cytoplasmwas derived from the newly collected wild-type stock to minimize any impact fromtransposable elements.The simultaneousexpression ofthetwo markersproducesyelloweye This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions 1144 W. R. RICE AND G. W. SALT color when the fliesare reared on standard medium. When kynurenineis added to the medium the vermilionmutationis not expressedand theeye color is brown(see Phillips, 1970). To measure philopatryfemales from habitat 4L were cultured onto standard medium and femalesfromhabitat 5E (1 g/liter) onto kynurenine-supplemented medium.Offspring derivedfromhabitatSE were thereforebrown eyed and those from 4L were yellow eyed. The v/ras phenocopy system provided a nonheritable genetic markerthatlabeled each flywithits habitat of origin. The markersystempermittedthe measurementof the degreeof resourcespecialization (philopatry)and also thecomponent responses to the disruptive selection for habitat preference.Habitat specialization was measured by recordingthe percentage to each ofthetwo brown-eyedfliesreturning selected habitats. It is only these fliesthat successfullylocated a suitable resourceand therefore potentiallycontributedgametesto the nextgeneration.On occasion, especially in the beginningof the experimentwhen habitat specialization had not yet evolved, the numberof flieschoosinga selectedhabitat was small and thereforethe percentage brown-eyedflieschoosing a habitat might be large or small owing to sampling error alone. To reduce this problem, whenever fewerthan 30 flies selected habitat SE or 4L, we conservativelypooled data from neighboringhabitats (i.e., habitats SE and 6E) so thata minimumtotalof 30 was used in calculating the percentage brown-eyed fliesin each selected habitat. The spatial and temporal responses to disruptiveselection were measured by recordingthe percentageof brownand yellow fliesthat chose all 24 habitats. Recall that (1) halfofthehabitatsare up and halfdown, (2) half are dark and half lighted,(3) half emanate the odor of ethanol (even-numbered habitats)and halfacetaldehyde,(4) a thirdare earlyand a thirdare late, and (5) choice on each habitat gradientcould be made independently.Changes in the phototaxis, geotaxis, chemotaxis, and development time of the subpopulations originatingin habitats SE (brown eyed) and 4L (yelloweyed) weremonitoredbycomparing thepercentageyellow-and brown-eyedflies choosing lightor dark,up or down, odd or even, and earlyor late habitats. Mating in Artificially Consolidated Groups The matingpatternsbetweenbrown-and yellow-eyedflies were measured intermittently(about every 5 generations)over the course of the 35 generationsof the experiment. In these generationsthe 60 mated females fromeach habitat were cultureda second time.Offspring fromthe second culturingswerecollectedas virginsand thesexes werestoredseparately.When 48 hrold they were placed, 25-yellowand 25-browneyed femaleswith 60-yellowand 60-browneyed males, into plexiglass matingchambers (6 cm radius and 2 cm high),and the matings were recorded visually for 1.5 hr. Parallel tests were conducted in lightedand darkened conditions.The numberof homotypic and heterotypicmatings were recorded. These testswill be referred to as the "forced consolidation matingtests." StatisticalAnalysis Changes in the components of habitat preferenceare displayed graphicallyin the Results section by simultaneouslyplotting data forthe fliesderived fromeach habitat (brown eyed from habitat SE and yellow eyed fromhabitat4L) vs. generationssince thebeginningof theexperiment.Significant differencesare indicated when 95% confidence intervalsforbrown-and yellow-eyed fliesconsistentlyfail to overlap over subsequentgenerations.To improveclarity,the 95% confidenceintervalsare not indicated on the graphs,but in all cases theyare no larger than ?5 percentage points. These small confidence intervals are a consequence of measuringa largenumberof flies (mean = 3,600 fliesper experimentpergeneration). In some cases the above criterionwas neverachieved,despitethefactthatthedata consistentlydeviated in the expecteddirection. To test the statisticalsignificanceof thesesmall but consistentdeviations,a contingencytest [the conditional binomial exact test (CBET, see Rice, 1988)] for2 x 2 tables withone or more small expectedcell frequencieswas used to compare experimental and control populations. The ana- This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions EVOLUTION OF REPRODUCTIVE lyzed data are Ai= ?//yellow-?brown i.e., the deviation between the phototactic(or geotactic,chemotactic,etc.)behaviorforbrownand yellow-eyedfliesin the ith generation. The firstcontingencyclassificationfactoris the sign of Ai (+ vs. -), and the second contingencyclassificationfactoris control vs. experimental.Only data forthe last half of the experimentwere considered in the contingencytests to permit ample opportunityfora response to selection to be realized. Changes in total habitat preferenceare measuredby plottingthepercentagebrowneyed flieswithinthe two selected habitats (SE and 4L) vs. generationssince the beginningof the experiment.In thiscase only a subset of the flies is analyzed, i.e., only those that successfullylocated one or the othersuitableresourceand thereforepotentiallycontributedgametes to the next generation. These smaller sample sizes yield larger 95% confidence intervals and the maximum 95% confidenceintervalis ? 18 percentagepoints. RESULTS Habitat specialization,as measuredbythe percentagebrown-eyedfliesin the two selected habitats,graduallyincreased in the two experimentalpopulations and did not develop in the control (Fig. 1). In the beginningof the experimentthe percentage brown-eyedflieswas similarin the two selected habitats in both experimentaltreatments. This, in combination with the data fromthe controlexperiment,indicatesthat the phenocopy systemdid not bias the experimentalresultswhen all habitat factors are considered collectively.By the end of the double-selection experimentno individuals were exchanged between the subpopulations utilizing habitats 4L and SE (i.e., 100% brown-eyedflies in SE, 100% yellow-eyedfliesin 4L). In the single-selection experiment habitat specialization evolved more slowlyand more erratically, but the outcome was virtuallyidentical to that of the double selection experiment (100% brown-eyedfliesin SE, 99% yelloweyed fliesin 4L, Fig. 1). No positive assortative mating, in the "forced consolidation mating tests," was observed at any time duringthe course of 1145 ISOLATION 1 00. 50 ,, 0 I 100 1 0 e 0--w 100 . ......... 50. 0 0 10 20 30 FIG. 1. The percentageof brown-eyedfliesin the two selectedhabitatsin relationto the numberof generations since the beginningof the experiment.Solid symbolsare forhabitat 5E (dark fruit)and open symbols forhabitat4L (white fruit).To remove variation in the total number of brown- and yellow-eyedflies thatemergedeach generation,numbersof fliesare expressedas the percentageofthe totalforeach eye color type.Upper, middle,and lowerpanels are the control, single-,and double-selectiontreatments,respectively. theexperimentin eitheroftheexperimental populations or in the control.Because the matingpatternsunderforcedconsolidation did not change over the course of the experiment,theywillnotbe discussedin detail here. The lack of development of positive assortativematingunderconsolidatedconditions implies that any prezygoticreproductive isolation evolving in the course of the experimentis solely a consequence of habitatspecialization.The reductionin gene flowis thereforeno greaterthanthatreflected by the divergencein percentagebrowneyed fliesshown in Figure 1. A direct translationbetween habitat selection data and gene flowrequiresthatthe fliesresolved themselves into the habitats firstand then mated locally. A preliminary experiment supported this assumption (Rice, 1985). The assumptionwas testedin the currentexperimentsin two ways. First, on rare occasions a collectionvial (habitat) This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions W. R. RICE AND G. W. SALT 1146 100_ 50 01 ~ .~I , . IV I , i 100-I 50- L bV VV I - 100 b VV.V ~ ~ ~ ~ ~~~~~~~~~ -D1 50 0 10 20 30 0 10 20 30 FIG. 2. The percentageof brown-(solid symbols)and yellow-eyed(open symbols) fliesthat selectedany of the 12 lightedhabitats(as opposed to the 12 darkenedhabitats).Upper, middle,and lowerpanels are thecontrol, single-,and double-selectiontreatments,respectively,data formales are on the left,femaleson the right. containingonlyfemaleswas observed.These weretestedbyexaminingthevials forlarvae 5 days later.In no case werelarvae detected. We also extensivelyobservedthe flieswithin the mazes. We never observed a mating anywherein a maze except occasionally at theentryportsto each habitat.Matingswere commonly observed within the habitats themselves. These observationsindicatethattherewas complete positive assortative mating between habitat preferencephenotypeswith respectto phototaxis,geotaxis, and development time,but incomplete,thoughsubstantial,positiveassortativematingwithrespect to chemotaxis. To investigate the impact of matingsbetweenhabitats3L and 4L and between habitats 5E and 6E, the data forthesehabitatswerepooled and Figure 1 was recalculated: no change in the pattern of habitat specialization was observed,nor was the degreeof breakdownin interhabitat migration diminished. We therefore conclude thatthesmall percentage of matingsthatoccurredat the entryports, separating adjacent habitats, had no importantimpact on our study. While therewere no detailed studies undertakento determinethe durationof time betweeneclosion of the fliesand theirsubsequent selection of one of the 24 spatiotemporalhabitats,a roughestimatewas obtained by noting when flies were first observedin thecentraltygontubes,and then when theywere firstobserved in the eight spatial habitats. Flies were routinelyobservedin the centraltygontubes on the day beforetheywereobservedin anyoftheeight habitats. Another indication that the flies spent many hours assortingthemselvesin the mazes is the observationthatwhen flies were counted immediatelyaftercollection froma habitat,flieswithunexpandedwings, lightbody color,or a visible remnantof the larval gut (indicatingthat they were only severalhoursold) werenot observed.These observations in combination with the fact thatmatingswere occasionally observed at the entryports leading to the habitats (D. melanogasterare not known to mate when less than 8 hr old) suggestto us that most fliesremained in the maze forat least half a day beforechosinga habitat. The factorsleading to habitat specializa- This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions EVOLUTION OF REPRODUCTIVE 1147 ISOLATION 100 50 / 0 0 , . o. *l ,~ , , , .~ . , 0 . . . . . 100 50 o O.Cl. a,.; 10 10 50 '' 0102030 30 20 .43 100 0 10 2~~~~~~0 3l.6 \ 0 0 1 ao Cl 0 FIG. 3. The percentageof brown-(solid symbols) and yellow-eyed(open symbols) fliesthat selectedany of the 12 upper habitats(as opposed to the 12 lower habitats).Panel arrangementis as in Figure 2. tioncan be resolvedby lookingat each hab- overlapping95% confidenceintervalcriteitatgradientseparately.Recall thatthemaz- rion forboth sexes). The geotacticbehavior ofthecontrolflies es resolvedthefliesin an orthogonalmanner so thata choice concerningeach of the four was affectedby the phenocopy technique habitatgradientscould occur independent- with brown-eyedflies choosing an upper habitat somewhat more oftenthan yellowly. Phototacticbehavior in the controlvar- eyed flies(binomial test,P < 0.0001; Fig. ied over the course of the experiment(Fig. 3). This bias in the behavior of the two eye 2) but this variationwas virtuallyidentical color morphs was in a directionthat parfor both brown- and yellow-eyed flies. alleled the artificialselectionin both experBrown-eyedfliestended to choose lighted iments.As shownin thecontroland thefirst habitatsslightly,thoughhighlysignificantly fewgenerationsof the experiments(Fig. 1), (binomial tests,P < 0.001 forboth sexes in however,the impact of this small bias was the control),more frequentlythan yellow- negligiblewhen all habitatfactorsare comin geotacticbehavior eyed flies.This bias was in the opposite di- bined. The difference rection of the artificialselection, and this observed in the single-selectionand the effectis consistentwithdata fromprevious double-selection experiments was larger studies (i.e., that a reductionin brown eye thanin the control(CBET contingencytest, pigment acts to diminish positive photo- P < 0.02) and graduallyincreased over the taxis;Fingerman,1952; Bumet et al., 1968). course of the experiments(Pearson's corIn the single-selectionexperimenttherewas relationtests,P < 0.05 in all cases). a small, though significant(CBET continChemotaxischangedlittleover thecourse gencytests,P < 0.0007 forboth sexes), di- of the experimentin the control,with no vergence in the phototactic behavior of systematicdifferencebetween brown and brownand yellowflies.In the double-selec- yelloweyed flies(Fig. 4). There was a small, tion experimenta highdegreeof divergence but significant(CBET contingencytests,P developed in phototactic behavior (non- < 0.02 forboth experiments)divergencein This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions 1148 W. R. RICE AND G. W. SALT 100 T 504 0 100-_ 50i 0 100- I , I 501 O 0 i 10 20 I 30 , 0 l 10 20 I I 30 FIG. 4. The percentageof brown-(solid symbols)and yellow-eyed(open symbols)fliesthat selectedany of the 12 acetaldehydehabitats(as opposed to the 12 ethanol habitats).Panel arrangementis as in Figure 2. the chemotaxisof brown-and yellow-eyed femalesthat was observed in both the single- and the double-selectionexperiments, but no significant changein thechemotactic behavior of the males was observed. Development time fluctuatedover the course of the experimentin the control,but in a mannerthatwas virtuallyidenticalfor both eye color morphs (Figs. 5, 6). In both experimentaltreatments therewas a gradual and large divergence in the development time of brown-and yellow-eyedflies(Figs. 5, 6, nonoverlapping95% confidenceinterval criterionfor males and females in the single- and the double-selection experiments). A detailed analysis of any interactions among the components of habitat preference (phototaxis,geotaxis,etc.) will not be consideredhere.We do point out,however, that the components of habitat preference combined in a roughlymultiplicativefashion. As a consequence, the proportionof flieschoosing a specifichabitat can be estimated by the product of the appropriate preferencecomponents,e.g.,theproportion of brown-eyedflieschoosing habitat 4L is estimatedby (proportionphotopositive) x (proportion geopositive) x (proportion chemoethonol) x (proportionlate). In summary,habitat specialization developed to a high degree in both experimental groups but not in the control.This resultedin complete or nearlycompletereproductiveisolationdue to a gradualbreakdown in migrationbetween the subpopulations utilizing the two spatially and temporally separated habitats. Developmenttime,geotaxis,phototaxis,and chemotaxis all contributedto habitat specialization in both experimentalpopulations. DISCUSSION Twenty-fiveyears of theoreticalworkby many authorscollectivelysupportthe conclusion that the selection-recombination antagonism is the principal genetic constraintto sympatricspeciation via disruptive selection (Felsenstein, 1981). The operation of the selection-recombination antagonismis consistentwith the factthat the experimentaloutcome of Thoday and This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions EVOLUTION OF REPRODUCTIVE 1149 ISOLATION 100 0 50 - R2 < e < \S 100 50 100- 501 0 10 20 30 0 10 20 30 FIG. 5. The percentageof brown-(solid symbols) and yellow-eyed(open symbols)fliesthat selectedany of the 8 earlyhabitats(as opposed to the 16 laterhabitats).Panel arrangementis as in Figure2. Gibson (1962) has not proven to be repeatable (Thoday and Gibson, 1970; Scharloo, 1971). Theoretical work by Slatkin (1982) and Rice (1984) predictedthat the selection-recombination antagonismcan be eliminated when reproductive isolation evolves indirectlyas a correlatedcharacter, and thereforethat sympatric speciation should be feasiblethis context.The experiments reportedhere, as well as previous work (Patemiani, 1969; Rice, 1985), support this prediction.There are several criticisms, however,that mightbe directedat our experimentaldesign. First,one mightargue that the levels of selectionused in our experimentswere too strongand therefore thatour resultsare misleading. We would disagree. The selection on habitat preferencewas certainlystrong. A flynot locating one of the two selected habitats had zero fitness.In nature, however, this is exactlywhat is to be expected wheneversuitable habitats are widely separated in space and/or time. That is, any individual that does not finda suitable resourcewillnecessarilyperishand contribute nothingto the next generation.We suggest that whenever the environmentalconditions are appropriatefor the development of speciationvia habitatspecialization,that is when suitable habitatsare distantlyseparated in space and/ortime, natural selection is like to be as strongas the artificial selection that was applied in our experiments. The second typeof selectionwas directed against"habitat switching."This typeof selection occurs when (1) habitatshave idiosyncraticselection regimes,and (2) adaptationto one environmentis at the expense of adaptation to the other. While there is evidence forthis typeof "trade-off'in nature(e.g., Vai, 1984; Rausher, 1984), itsfrequency and magnitudeare still unknown. To bracketthe level of habitat-specificselection thatmightbe realized in nature,we used two levels of selection:(1) no habitatspecificselectionin the single-selectionexperiment,and (2) very stronghabitat-specific selection in the double selection experiment.Since the outcome of both experimentswere virtuallyidentical,we con- This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions W. R. RICE AND G. W. SALT 1150 100-. 50-= . 100- 100- 50 N: 50 0 / _ 0 __ __ 10 ,- ,, _ 20 30 0 10 20 30 FIG. 6. The percentageof brown-(solid symbols) and yellow-eyed(open symbols)fliesthat selectedany of the 8 late habitats(as opposed to the 16 earlierhabitats).Panel arrangementis as in Figure2. clude that selectionagainst habitat switch- migrationpattern,sexual signaling,deil acing, while not necessary,can nonetheless tivitypattern,and the patternof reproducstronglyreinforcedisruptive selection in tive activityduringtheannual cycle(fordisgenerating reproductive isolation under cussion see Rice, 1987). sympatricconditions. As a thirdcriticism,one mightarguethat A second criticismof our experimental speciation has not occurredin these experdesign might be that the environmental imentssince reproductiveisolation was abcontext simulated in the experimentswill sent outside the contextof the mazes and rarelybe realized in nature,and therefore was not shown to be mediated by mate this form of sympatric speciation, while choice or reducedhybridviability.Certainpossible, has verylimitedapplication. This ly we have not produced entitiesas distinct is an ecological ratherthana geneticalprob- as horses and donkeys. As pointed out by lem. A discussionofvarious naturalhistory Mayr(1963), however,populationsneed not contexts,which can lead to strongdisrup- produce a sterileF1 to be considered sepative selectionon habitatpreference,can be rate species. All thatis requiredis indepenfoundin Rice (1987). Here we pointout that dentgene pools when populationsare withwhile our experimentsspecificallysimulat- in the"cruisingrange"ofone another.Mayr ed disruptive selection on habitat prefer- also pointsout thatit is common forsibling ence, the experimentswere designedto ad- species to occasionallyhybridize,especially dress the more generalcase of reproductive under unnatural conditions (such as our isolation evolvingindirectlyas a correlated forcedconsolidation experiments). While we agree that isolation via mate character.Wheneverthis occurs, the selection-recombination antagonism is elimi- choice and hybridinviabilityare hallmarks nated and sympatricspeciation is geneti- of extantspecies pairs,we do not agreethat cally feasible.Other formsof selectionthat theseneed be manifestin theearlystagesof might lead to reproductiveisolation as a speciation. We suggestthat only a biologicorrelatedcharacterinclude, for example, cally induced breakdown in gene flow(gedisruptiveselectionon body size, dispersal, netically controlled migrationdiminution This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions EVOLUTION OF REPRODUCTIVE in our case), which operates under natural conditions(the mazes in our case), must be established to demonstratethat speciation has occurred.What has evolved in our experimentsis the fissureof one gene pool, originallyunifiedby migration,into two independentlyevolvinggene pools. Although manyoftheattributesassociated withmore ancient species pairs are absent,the critical step in speciation, i.e., a nearly complete breakdown in gene flow, has indeed occurred.While we catoragizethepopulations utilizing habitats 5E and 4L as incipient species,we leave it to thereaderto conclude whetherspeciationper se has occurred.We should point out, however, that we would expectthepopulationsutilizinghabitats5E and 4L to eventuallyevolve, due to their isolated gene pools, additional isolation via mate choice and hybrid inviability via pleiotropy,as is postulated to occur in the allopatric model of speciation (Muller, 1942). A fourthpotentialcriticismconcernsthe question of whetherthe incipient speciation,thatwe believe to have occurredin our experiments,was truly sympatric,i.e., it mightbe argued that because the habitats are separated in space and time they are allopatricand all thatwe have done is simulated allopatric speciation (or at least its earlystages).We would disagree.Recall that in the beginningof the experimentsthere was freegene flow(migration)betweenthe subpopulations exploitinghabitats 5E and derived fromboth hab4L, since offspring itats were initiallyequally likelyto choose thetwo selectedhabitats(Fig. 1). By theend of the experimentgene flowwas absent,or nearlyso, in both experimentaltreatments. In our experimentsreproductiveisolation did not evolve becauseofan extrinsicbarrier to gene flow,as evidenced by the fact that no reproductiveisolation evolved in the control.Instead, reproductiveisolation evolved because of phenotypicchangesthat evolved in the fliesthatcaused them to assort themselves into different parts of the environment.Thus allopatry was not enforcedby an extrinsicbarrierto gene flow, but instead behavioral and development allopatryand changesevolved thatproduced led to reproductiveisolation. Clearlythisis not a case of allopatricspeciationsince migrationbetweenhabitats5E and 4L was not ISOLATION 1151 prevented,and did occurat a highrate(about 50%, see Fig. 1) in the control and during the early generationsof the experimental treatments. One of the patternsapparentin Figure 1, and in some cases in Figures2-6, is thatthe responseto selection,was slowerin theearly compared to the latergenerations.Our hypothesis forthis effectis thata formof advantageouslinkagedisequilibriummayhave been builtup in theearlygenerations,which permittedthe more rapid responseto selection in the later generations.Because we were selecting on four traits (presumably polygenic)at once, antagonisticlinkageassociationsbetweenpolygenesforthe different traitsmightretardthe earlyresponseto selection.As selectiongraduallybrokedown such antagonisticlinkageand thenbuilt up complementaryassociations, the response to selectionwould be expectedto accelerate. We have no evidence forthis scenario but offerit as an hypothesis. In conclusion, we thinkthat one of the principaldifficulties with the studyof speciation is that it occurs quite slowly on a microevolutionaryscale, despite its apparent rapidityin the fossil record. This fact complicates a directobservational investigation of the speciationprocess,and thereforemost inferencesabout speciation have been based on comparisonsbetweensibling species and comparisonsbetweenallopatric populations of the same species. If directobservationof speciationis precluded, then,how does one testthe hypothesis that sympatricspeciation has been an importantmechanismin thepropagationof new species? It seems to us that the only way to make progressis to use laboratory model systemsto determinewhethersympatricspeciationis biologicallyplausible. If thiscan be shown,it would seem reasonable to conclude that sympatricspeciation has probablyplayed a nontrivialrole as a speciation mechanism. To supportthe plausibilityof sympatric speciation two thingsmust be established: (1) thatthegeneticmechanismsrequiredfor the process to operate are feasible within the frameworkof population genetics,and (2) that the natural historycontexts promoting sympatric speciation are ecologically feasibleand prevalent.Previous work (e.g., Jaenike, 1985; Tauber and Tauber, This content downloaded from 128.171.57.189 on Wed, 25 Mar 2015 19:26:39 UTC All use subject to JSTOR Terms and Conditions W.R. RICE AND G. W. SALT 1152 1977; Bush and Howard, 1986; Rice 1987) suggeststhatmanynaturalhistorycontexts, whichpromotebothdisruptiveselectionand the evolution of reproductiveisolation as a correlatedcharacter,are common. 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