1. No category

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
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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
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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.
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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
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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
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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-
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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)
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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-
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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
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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
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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-
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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
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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,
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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. The experimentsdescribed here, in combination
with the plant work by Paterniani (1969),
supportthe conclusion that sympatricspeciation is geneticallyfeasible wheneverreproductiveisolation evolves indirectlyas a
correlatedcharacter.We concludethatwhile
most speciationmay take place in allopatry
and conformwith the allopatryparadigm,
sympatricspeciation is an importantand
viable alternative.
ACKNOWLEDGMENTS
We thankLarryHarshman forproviding
the stock of fliesused to begin the experiments,B. Dennis forillustratinga habitat
maze, S. Andrews,J. Kim, B. Kooper, D.
Oliveras, G. Stryker,and S. Sivinski for
technicalassistance, and K. Ono forcomments on the manuscript.This work was
supportedby GrantBSR 8407440 fromthe
National Science Foundation.
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CorrespondingEditor: J. M. Ringo
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