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On the maintenance of allozyme and inversion polymorphisms in Drosophila
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Kamping, Albert
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Kamping, A. (2000). On the maintenance of allozyme and inversion polymorphisms in Drosophila
melanogaster: Interactions between Adh, aGpdh and In(2L)t Groningen: s.n.
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Chapter7
Summarizingdiscussion
Most animaland plant taxa possess
high levelsof geneticvariation.Within specics,often
considcrabledifferentiation for various genetically determined traits exists between
populationsfrom diffcrent geographicorigins.A major questionin evolutionarygeneticsis
whether these differences in genetic constitution reflect adaptation to pafticular
environmentalconditions,are merelydue to chanceprocesses
or havea historicbackground.
Whennaturalselectionis responsiblefor geneticdifferentiationit canbe askedat which level
uaturalselectionoccurs.As many geneticallyvariabletiaits show similar correlationsrvith
environmentalgradients,selectionon a particularpolymorphismcan lead to gcneticchanges
in functionallyand/orgeneticallylinkedtraits.
In this thesisthe resultsof a studyon the adaptivevalueand evolutionof somegenetic
polymorphismstn Drosophilamelanogasle/'are
presented.
It is tried to causallyrelategenetic
constitutionwith environmentalconditions.In this respect,the functionaland chromosomal
interactions of two protein coding genes, alcohol dehydrogenase(Ádh) and cr,glycerophosphate
(aGpdh), and a chromosomalinversion fln(2L)tl, are
dehydrogenase
investigated.The tluee polyrnorphismsare all locatcd on the lcft arm of the second
chtomosomc.
ht(2L)t includesthe a{ipdh gene,while theAdh geneis locatedjust outsidethis
inversion. Almost all natural populations of D. nelanogaster contain two common
electrophoretically
distinguishablealleles both for Adh (Adhs and AtlhF) and for aGpdh
(aGpdhs and aGpdhF).In(2L)t is nearly always associatedwith the Adhs and,the aGpdhF
alleles, while Stanclard(57) chromosomescarry all possible combinationsof alleles.
includingAdhs/aGpdht'.The lrequencyof .ldht increascsfrom around0. l0 in populations
from temperateclimatesto above0.90 in tropicalregions.aGpdhFfrequenciesrangefiom
about0.50 in temperateregionsto above0.90 in the tropics.In(2L)t frequencies
rangefrom
lessthan 0.05 in populationsfrom temperateclimatesto around0.50, lvith extremesup to
0.70,in the tropics(references
in Van Deldenand Kamping 1989, 1997 Kampingand Van
Delden1999a;Van't Landet a|.2000).
Geneticfluctuations in a seminaturalpopulation of D. melanogaster
Adh and aGpdh allele frequenciesstronglyflr"rctuated
over a pcriod of 25 years(i.e. about
populationof D. melattogasler
400generations)
in a seminatural
kept in a tropicalgreenhouse
(chapter 2). Many short-tetmand a few long-termfluctuationsoccurredfor both genesin
this period.The pattemof the fluctuationsu,asnot random:comparisonof allele frequencies
betweenseasonsshowed significantly hrgherAdhs and aGpdhtsfrequenciesin summers
comparedto winters. This svstematicdifferenceamong seasonsis clear evidencefor the
actionof natural selection:i.e. differencesin fitnessbetweengenotypesin relation r,r,ith
environmentaldifferencesamong seasons.
Apart from their significantpositive correlation
rrith environmentaltcmperalure.Alhs and aCpdhFallele fi'equenciesare positively
correlated.
The observednon-randomassociationof alleles(gameticdisequilibriunr)of thc
two gencsin many samplescould indicateepistaticinteractionbetweenthe two loci. Gametic
Chapter7
is positivelycorrelatedwith ALlhs'aGprlÍ'haplotype
disequilibrium
frequencyand alsowith
erlvironmentaltemperature.
As hoth allele combirrationsAdh\/aGp,lltrandAlhFraGpdhsare
in excessin the caseof increaseas well as decrease
of Atlhs and acpdhF allele frequencies,
it
seemsunlikely that selectionon the allozyme polymorphismsis the main cause for the
with the Adhsand
observedchanges.The chromosomalinversionIn(JL)t, which is associated
aGpdh' allele combination,appearsto be largely responsiblefor fluctuationsin allele
frequencies
aÍ ÍheAdh andaGpdhloci. In fact,the allozymeloci arehitchhikingwith In(2L)t.
Also deviationsfrom the expectedAdh genotypicratiosin somesamplescanbe explainedby
selectionat the chrornosome
level.It is convincinglyshorvnthat naturalselectionis opcrating
on In(2L)t polyrnorphism,u'hich has a large impact on the allozyme polymorphismsby
association.
cluomosomal
Changes in environmentalternperaturewill intluence various factors, like egg
productionandlan,al density.Therefore,intraspecificcompetitionfor food and differences
in
genotypes
probably
rate
karyotypcs
important
fitness
among
and
are
developnrental
in naluralD. melunogaslerpopulations.Such tcmpeÍature-related
characters
may
characters
be responsiblefor the obsen'edfluctuationsin genetic composition of the greenhouse
population. No sharp distinction can be made between functional and chromosomal
of the Ádlt and aGpdh loci as causefor the non-randomassociationof alleles
associations
observedin this population.Moreover,non-randomassociationofalleles observedin natural
D. melanogaslerpopulationsmay also be causedby severalother factorslike geneticdrift,
subdividedpopulations,migrationand non-randommating.
Geneticchangesin D. melanogaslerpopulationswith different geneticconstitution
A logical next step was to separatethe effects of environmentalstresson eachof the
polymorphismsand to establishhor.vinteractionsthrough functional and/or chromosomal
linkageoccur (chapter 3 antl chapter 4). For this purposeexperimentalpopulationsrvith
and the geneticcomposition
differentallozymeand karyotypeconstitutionwere constructed,
was followed under various environmentalconditionsin the courseof time. The InQL)t
polymoryhismwas studied in a geneticbackgroundin rvhich either none, one or both
allozynreloci were polynrorphic.On the other hand, each allozyme polymorphisÍnwas
studiedin the presenceor absenceofvariation at the otherallozymelocusandin thepresence
of inversionpolynoryhism.This allorvcdthe analysisof multigenicreactions
on
or absence
basisof monogenicreactions.Chapter 3 dealswith eflèctsof variousrearingtempcratures
on the allozymeand.In(2L)t polymorphismswhile in chapter 4 effectsof populationdensity,
developnrenttime arrd environmentalethanolare presented.The environmentalconditions
were chosenbecauseof thcir presumcdreler,ancewith respectto natural habitatsof D.
The allcle and karyotypefrequencydataclearly shou'that naturalselection
melanogaster.
is
acting on eachof the polynrorphisms.
but the dircction and magnitudehighly depends
on
geneticbackgroundof the otherpolyrnoryhismsand on environmentalconditions.Themost
relevantfindinssu'ill bc summarized.
r34
Summarizingdiscussion
In(2L)tfrequencies
An overallstrongdisadvantage
of In(2L)t was observedin experimentalpopulationsat lower
temperatures,
at high larval densitieswith 14 days generationinterval (HDl4), and at food
supplcmentedwith
ethanol.At29.5",33oandat high larvaldensities
with 2l daysgeneration
rnterval(HD21), In(2L)t frequenciesrverc highestbut not significantlydcviating from the
initial frequencyof 0.50. Frequencies
in the latter environmentsagreewith averageIn(2L)t
frequencies
in populationsfrom tropical regions(referencesin Van Delden and Kamping
1991;van't Land 1997;veuille et al. 1998;van't Land et at.2000).The observed
In(2L)t
frequencies
in the variousenvirorunents
areconcordantwith the highersurvivalratesat high
tenrperature
and the longer developmenttinre ol In(2L)t homokaryotypes(Van Delden and
Kamping 1989, 1991). In(2L)t apparentlycontainsgenetic variants for slower juvenile
development,
and comparedwith control conditionsal 25a, the specific allelic content
providesselectiveadvantageat higlt temperatureand disadvantage
at lolv temperatureand
ethanol-richenvironments.At extremely high temperature,significant superiority of
In(2L)t/sr heterokaryotypeswas observed: the advantageous effect in In(2L)t
homokaryotypes
is probablycounteracted
by geneswith deleteriousel-fectsor by a general
effectof homozygosityof the In(2L)r region (about 15% of the genome) as In(2L)t
anangements
areexpectedto be geneticallyuniformto a largeextent(chapter 6).
Ádhwul aGpdh allelef'equencies
In monomorphicSZ populationskept at different temperatures,
larger departuresfrom the
initial allele frequencyof 0.50 were observedfor both Adh and aGpdh in populations
segregating
for one of the loci, comparedwith populationssegregatingfor both loci.
However,significantdifferencesamong temperatureswere absent rvithin each of these
groups.The larger departureol Adhs from the initial frequencyof 0.50 cornparedw,ith
aGpdht,agreeswith lower Adhs than aGpdhs frequenciesin D. melanogaslerpopulations
fromregionswith a temperateclimate(Parkashand Shamina1994;Bubli et at.l996; Yan 't
Land1997).
Individualscarrying the dipdhF allele showed faster developmentunder high-density
conditions.This agreeswith frequencyfluctuationsobservedin the seminaturalpopulation
(chapter2; Kamping and Van Delden 1999a)and experimentaldata by Maàrl<ovic et al.
(1987).High-densifyconditionsmay resemblethe tropical situationwith high numbersof
individuals
andhigh dlpdhF frequencies,whL'reas
at higherlatitudes,with slowerdevelopment,
individuals
carrying the aGpdhsallele probablyhave a relative advantageas comparedto the
tropicalsituation.Populationsffom tenrperate
regionswill haveperiodswith low population
sizes
because
of foodshortage
andunfavourable
weatherconditions.Geneticdrift andmigration
will be impoftantin thosepopulations.
Theseextremeenvironmental
conditionsare generally
rvitha decrease
associated
in rnetabolicrate,rvhichimpliestliat selectionmay occurat the level
of energycarriers.The higher flight ability of cíJpdhssgenotlpes, through a better energy
supplyof the aGPDHssallozl.rncto flight musclesat low temperature(Bames and LaurieAhlberg
1986),maybe relevantin thisrespect.
The higherAdhF and dlpdhs allele frequencieson food supplernentedu,ith ethanol
compared
to controlfood agreeswith earlierÍindings(Cavenerand Clegg 1981;Van Del<len
4É
I35
Chapter7
and Kamping 1989). Diffèrcntiationin Adh and aGpdh allele frequenciesbetween D.
populationsfrom rvineriesandtheirsurroundings
(e.g.Hickey andMclean 1980;
melanogaster
Alonso-Moragaand Mufloz-Serrano
1986)may be explained(at leastpartly) by the strongly
reducedfrtnessof In(2L)/ karyotypesin the presenceof ethanol,which nreansthat selective
effectson theallozyne loci couldhavebeenoverestimated
by ignoringIn(2L)t polymorphism.
The large differentiationof Ádh as well as d]pdh allele frequenciesdue to different
densityconditionsand the presence
of ethanolcomparedto the effectof varioustempeÍatures
shorvs that the role of the former environmentalfactors is more irnportant for genetic
with optimal
diffelentiationattheÁdh andaGpdhloci thanthe effectsof varioustemperatures
loodcondilions
tTablcI)
in experimental
Tablc 1. Summaryof the changesin In(2L)t,Adhs nd aGpdhFfrequencres
populatrons
with initialfiequencies
of 0.50.
Environment
200
25" (Control)
29.5"
33"
HD14
HDz]'
Ethanol
In(2L)t
Adhs
aGpdht'
0*
0*
0
0
0
0
0
0
0
0
0
+
Populationswere kept under optimal food conditionsat four different temperaturesand underhigh
larval densrty conditions with transfer times of 14 and 2l days (HD 14 and I{D 21), and food
supplementedwith ethanolat 25o. For In(2L)t, the Adh and aGpdh backgroundsare combined.For
Adh and aGpdh only Standord chromosomes are involved, while the aGpdh respective\yÁdh
backgroundsarecombrnedin this overview.-: significantdecÍeasein tiequency;0 : minor changes
in frequency;+: sig'nificanÍincreasein frequency;* : overdominance.
Interactions befli,een tlte pohmorphisms
The experimental populations polymorphic for both Adh an<ldipdh, either polymorphic ior
In(2L)t and SZ or monomoryhic for ,S|, were startcd with maximum ganretic disequilibrium
between Íhe Adh and aGpdh loci. Initially only Atlhs/dlpdhF and Adh'/cfrptlh'gametes were
present. Tlre other trvo gametcs (4(tht /aGpdhF and, ,ldhsiaGpdhs) appeared with time,
depending on the number of generations,recombination fraction betwecn the Adh and aGpdh
Ioci and fitness differenccs between Adhl dlpdh
genotypes. In experimental populations
varying for all three polymorphisms, the initial gametic disequilibrium between Adh and
discussion
Summarizrng
ffipdh alleleswas maintainedunderall environmentalconditions(chapter 3 and 4) due to
the stronglysuppressed
recombinationin h(2L)t/ST heterokaryotypes.
The consequence
of
thehigh and stablegameticdisequilibriumis a correlatedresponsein Adh and aGpdh allele
frequencies.
Thc trvo allozymegenesand the In(2L)t polymorphismgeneticallybehaveas
gene,
one
rvith three genotypes.Hornozygotesand heterozygotesare homozygousor
heterozygous,
respectively,for all threepolymorphisrns
andprobablyfor many othcrgenesas
(chapter 6). Changesin allele frequenciesat the
h(2L)t andSIare geneticallydifferentiated
Adh locusin this populationtype are associated
with a changeat ïhe aGpdh locus and vice
versa,and are in fact govemed by changesin In(2L)t frequencies.Observedgametic
disequilibriaamong ïhe Adh and aGpdh loci in this populationtype are due to hitchhiking
wiïh In(2L)t and the magnitudeof the hitchlliking effect is nearlymaximal. The correlated
responsein Adh and aGpdh allele frequencieswas also observed in the seminatural
greenhouse
populationwhich was follorvedfor many years(chapter 2; Kamping and Van
Delden1999a).
ln In(2L)t-treepopulations,gameticdisequilibriumbetweenAdh and atiptlh decreased
with numberof generations.The faster decay of gametic disequilibriumat high rearing
temperature
comparedto low rearing temperatureis causedby a significantly higher
recombination
rate at high temperature.The observedincreasein recombinationrate in
stressis consistentwith a generalaccelcrationof evolutionary
response
to high-temperature
changeunder extreme environmentalconditions (Parsons 1988). Under high-density
with ethanol,the rate of decayof gameticequilibrir.un
conditions
and on food supplemented
was influencedby epistatic interactionsbetweenAdh and aGpdh. Fitness interactions
betiveen
Atlh/ffipdh two-locusgenotypesunderethanoland high-densitystressare ascribed
to thefunctionalrelationshipof the two allozymepolymorphisms(e.g.McKechnieand Geer
1988;Oudmanet al. 1994). Gametic equilibrium values were ultimately reachedin all
environments
in In(2L)t-free populations,though after varying numbers of generations.
populationsseem
Gametic
disequilibriabetweentheseloci observedin wild D. ntelanogasler
of the presenceof
to be causcdby reducedrecombinationfrequencies.as a consequence
ht(2L)t,ratherthanby epistaticinteractionbetweenthe loci.
in In(2L)t frequenciesrvere dependenton the constitutionaI Íhe Adh and
Charrges
ctGpdh
Ioci.In general,populationspolynrorphicfor Adh had lou'et hr(2L)t frequencicsthan
monornorphic
ldlss populations,rvhile populationspolymorphicfor diptlh exhibitedhigher
than mononiorphicaGpdhFrpopulations:the selectivedisadvantage
for
In(2L)tfrequencies
In(2L)tat low temperaturcsis reinforcedby the Adh polymorphismand reducedby the
aGpdh polymorphism.At high temperature,populations monomorphic for Adhss and.
ffiptlhFF,exhibitedhigher In(2L)/ frequenciesthan populationspolymorphtc for Adh andlor
fixed or closcto fixation for Adhs and aGpdhFresemblenaturaltropical
acptlh.Populations
populations
whereIn(2L)t frequenciesare relativelyhigh (e.g.Andersonet al. 1987;Yan'I
Van 't Landet aL.2000\.
Land1997;Van 't Landet a1.1999'.
Experimentalpopulations polymorphic for In(2L)t exhibited in most casesa depressing
effectof In(2L)t on Adhs and cíipdhF flrcquencies:strongest cffect was observed on ethanol
supplemented
food and under high density with two-rveeks generation interval (disadvantage
for slowly developing individuals) and the effect diminished with incrcasiug temperature
t37
Chapter7
while at 33" cvena reversalwas observed.The negativeeffectsof In(2L)/ at low temperatures
are strongerwhen aGpdh is polynrorphicand also strongertvhen Adh is monomorphic.
Differencesin relativefitnessof fu(2L)t andSIkaryotypes,dependingon geneticconstitution
of ïhe Adh and ffipdh genes,emphasise
the role of the geneticbackgroundin selectionon
particularpolymorphisms.
Selectionpressureon the threepolymorphisns
Selectioncoefficients
calculated
frornthechangesin In(2L)t frequencies
andassumingselective
disadvantage for In(2L)tlIn(2L)t homokaryotypes only, yielded high values in some
environments.For example,at 20", at high larval density with 14 days generationinterval
(providing disadvantagefor slowly developinggenotypes)and at ethanol supplementedfood,
selectioncoefficientsdeducedfrom ffequencychangeswere 0.47,0.51 and 0.83,respectively,
in expcrimentalpopulations monomorphic for Adhss and aGpdhFF.The assumptionthat
selectionis actingexclusivelyagahslIn(2L1tlh(2L)thorlokaryotypes
is basedon differences
in
developmental
time betweenkaryotlpes in theseenvironments(Van Delden and Kamping
1991; Van't Land 1997).The two high+emperature
environmentsare characterised
by a
significiurtexcassof heterokaryotypes
in many samples,indicatingoverdominance
underthese
conditions.The observedexcessof heterokaryotypes
agreesrvith higher fitnessvaluesof
heterokaryotypesfor some fitness traits under high{emperaturcconditions (Van Delden and
Kanrping1991,1997,KampingandVan Delden1999b).
The much higher differentiationamong environmentsfor the In(2L)t polymorphismas
comparedto the two allozymepolymorphisrnspoints to strongerselectionpressureson In(2L)t
in extremeenvironments.Selectioncoefficientscalculatedfiom ffequencychangesin the
experimentalpopulationsand assumingselectionagainstone of the homozygotes,do not
exceed0.05 for ïhe Adh and aGpdh polyrnorphismsassociatedwith ,tI arrangements
(except
for Adh on ethanolmedium,where the selectioncoeffrcientagainstAdhssis 0.18),while
selectioncoefficientsfor tn(2L)tllnQL)r homokaryotypesreachup to ten fold higher valuesin
extreme environments.Therefore,genetic divergenceamong, and temporal variation rvithin
populations
naturalD. melanogasler
seemsto be moreinfluencedby seleclionon chromosomal
variation than on allozymevariation. Tlic ratio of Adhs/acpdhÁ haplotypesassociatedwith
In(2L)t and SZ will determine the magnitude of fluctuations in Ádh and ffipdh allele
frequencies
in naturalpopulations.
Fitnessdifferencesbetweenkaryotypesunder high-temperatureconditions
The causalrelationbetweenÍhe In(2L)t polymorphisrnand high-temperature
resistance
was
studiedfor some fitnesscomponentsin chapter 5. Sterility in both sexeslvas inducedby
juveniles as wcll as adultsto 33ofor
exposingIn(2L)t and SIhomo- and heterokaryotype
sonretime. Fertility can be restoredafter a recoveryperiod al 25". Fertility restoration
in
In(2L)t homo- and hetcrokaryotypes
was significantlyhigherthan in SI homokaryot)?es
for
both sexesand heterokaryotype
superioritywas positively correlatedwith severityof the
high-temperaturcstress.A genetic increasein ferlility restorationwas observedfor all
karyotypesafter I 0 generationsselectionat 33' (includinga recoveryperiod at 25obefore
startinga new generation),
indicatingthe presenceofgeneticvariationfor fertilityrestoration.
ll8
Chapter7
increasingdifferencesin relative fitncssbetweenhomokaryotlpeswith increasinglatitude
(Fig. 1). In caseof heterokaryotype
superiority,and assumingdifferentratiosof the selection
coefficientsagainstthe two homokaryotypes,
In(2L)t rvill reach equilibrium frequenciesas
shown in Figure l. Theseequilibrium frequenciesare only dependenton the ratio of the
selectioncoefficientsand cannot provide any information on the intensity of selection.
However,the numberof generations
neededto reachthe equilibriumfrequencywill increase
with lor"'erabsolutevaiuesof the selectioncoefficients.
1.00
0.80
o
o)
f,
c)
0.60
L
.f,
0.40
=
f
o
tiJ
0.20
0.00
46
Ratio of selectioncoefficients(s/t)
Figure 1. The relationbetweenthe ratio of selectioncoefficients(s and /) againstthe trvo
andIn(2L)tequilibriumfrequencies
homokaryotypes
underthe overdominance
model.Fitncsses
of
and.SI/SIare: 1-s,I and1-r,respectively.
In(2L)t/ln(2Ltt,In(2L)t/ST
Data concerningthe relationshipbetweenallozymeheterozygosityat single loci and
fitness or Íitness-relatedcharactersin optinral and slressful environmentsare scarce.
However, it is often assumcdthat allozyme polymorphismsare maintainedby a higher
(referencesin Van Delden 1982 and Chambers1988).
overall fitnessof the heterozygotes
Without disturbing effects of In(2L)t, an excessof heterozygotesfor both single and
combinedallozymeloci was observedat high temperature
and underhigh-dcnsityconditions
in more samplesthanexpectcd.This indicatesa role for balancingselectiondirectlyactingon
thc allozymeloci in maintainingthesepolymorphisms.As shown in chapter 3 and 4, the
relativefitnessesof Adh as well as aGpdh genot)?esalso dependon geneticconstitution
of
the other locus. The observedpositive relationshipbetween severity of environmental
at the Adh and dlpdh loci, however.is mainly
conditionsand excessof heterozygotes
explainedby associative
overdominancc
throughthe hitchhikingeffect wilh h(2L)r (chapter
2. 3 and4).
140
Summarizingdisoussron
With respectto theAdh, aGpdh andh(2L)t polymorphismsfifteenmultiplc genotypes
canbe distinguished(Table2). Four of thesegenotypesareIil(2L)í/STheterokaryotypes:
all
wrth Adhs/aGpdÀrassociatedwith the In(lL)t chromosomc,bu1 each with a different
Adh/acpdh allele cornbination(FF, FS, 5,F or S,Í) on the 5I chromosone. The four
In(2L)t/ST genotypesrnay exhibit different fitnesses,through interactionsbetween the
alloz).,rneand In(2L)t polymorphisms, and the fitness relations may differ among
enviroumental
conditions.
Table 2. Adh/dlpdlt allele combinationsibr Standardand In(2L)t chromosomes
and the
genotypes.
corresponding
In(2L)í
Standard
Haplotypes
FF
rs
sr'
,ts
s.F1
FF
FF
rs
^9F
,s,s
.tr'1
FH
HF
HH
HF ST/I
FS
HH
,SF
HS
SH
HH ST/I
SF ST/I
SH ST/I
SF I/I
,tó'
1:
indicatethe Adh and dlpdh allele,respectively;
Thefirst and secondletterof the haplotypes
Genofypes
for the
no chromosome
indicationmeansStandanlchromosome.
In(2L)tclromosome;
.Fast
Adhand&pdh polymorphisms
areindicatedby F, ,t or É/,rvhereF andS indicatehomozygote
ST/I: heterokaryotype
Standard/In(2L)t;
I/Il
respectrvely,
and É1standsfor heterozygoï.e.
andS1orv,
indication:homokaryotype
Standard/Stundard.
homokaryotype
In(2L)t/In(2L)r;no chromosome
Some relevant examplesof such heterokaryotypesuperiority with varying fitness
with different.4dh/aGpdhgenotypesaregivenin Table3.
relaïions
betweenheterokaryotypes
All SI andIn(2L)t homozygotcsare assuntedto have similar htnesses.Thcseexampleslead
to "equilibrium"frequencies
which are ratherunusualfor naturalpopulations,but show that
the impact on the allozyme polymorphisms depends on fitness relation between
with differentAdh/aGpdh genot)?es.The equilibrium frequencieswhich
heterokaryotypes
superiority(Tablc 3A and 38) arc in fact the starting
arethe result of heterokar-rotype
populationswhich are follo$'edfor their geneticcomposition
lrequencies
of the experirnental
underdifferentenvironmentalconditions(chapter3 and4). Furtherinvestigationleamedthat
which are heterozygoustbr Adh (Table
superiority
of the two In(2L)t/STheterokaryotypes
ol AdhsandInl2L)t and maximumgametic
3C.example3) leadsto a complcteassociation
disequilibrium
betweenAdh and ffipdh as observedin D. melanogasterpopulalionsfrom
Meditenanean
climates. Superiority of the trvo In(2L1t/,9Theterokaryotypesrvhich are
ol aGpdhnand
for aGpdh (Table3C, example4) leadsto cornpletcassociatiot.t
heterozygous
populations.
found
D.
nelanogaster
in natural
In(2L)t,buÍis never
4
Chapter7
In order to explain the influence of In(2L)t polymorphism on the geographic
distributionpattem of the Adh and ffiptlh polymorphismsin tropical, Mediterraneanand
temperateregions, simulationswere performedby assumingfihress differencesbetrveen
ht(2L)t and ,SZhomo- and heterokaryotypes
only (Table4). It shouldbe noted that the Ádh
and aGpdh allele frequenciesfound in tropical regionsand Mediterraneanclimatescan be
explainedfully by overdominanccof In(2L)tiSI and assumingvarying fitnessrelationships
betwcenheterokaryotypes
with differentAdh/aGpdhconstitution.
Table 3. Fitness differences among In(2L)t/ST heterokaryotypes with dilÍerent Adh/d;pdh
genollpes and its consequencesfor lhe Ádh and a{ipdh polymorphlsms (see text for further
exolanation).
Fitness v aluesIn(2 L)I/ST
sln
Equilibrium frequencies
,s11
HF
HH
AdhS
1.0
t.2
1.0
1.0
1.0
1.0
1.2
1.0
1.0
1.0
1.0
1.2
1.2
1.0
t.2
1.2
\.2
1.2
1.0
1.2
t.0
1.2
1.0
t.2
1.0
1.2
1.2
1.0
D/D^^
aGpdh'
In(2L)t
1.00
1.00
0.50
0.50
1.00
0.50
1.00
0.s0
0.50
0.50
0.50
0.50
t.2
1.2
t.2
1.0
0.50
0.50
1.00
L00
0.50
1.00
0.50
1.00
0.50
0.50
0.50
0.50
1.00
1.0
t.2
t.2
t.2
0.50
u ./ )
0.50
0 . 75
0.50
0.15
0.75
0.50
0.00
0.50
0.50
0.50
0.00
0.14
1.00
i.00
A:
1.2
1.0
1.0
1.0
B:
1.0
1.2
1.2
1.2
C:
1.0
t.2
1.0
1.0
1.00
Various fitnessrelationsbetweenthe four In(2L)t/ST genotypes(see Table 2) are shown,with the
correspondingequilibrrum frcquenciesfor Adh, a{}pdh and h(2l)t, and gametic disequilibrium
values (D/D^.,) between Ádh and a{}pdh. F-itnessvalues for all other genoqpes are ad.;usted
to 1.
Initial frequenciesof the data presented:
.4dhs - 0.50: cíJpdht : 0.501In(2L)t < 0.01.A: One
In(2L)I/ST genotypewith arbitrarily chosenhigher fitness value than all other l4 genotypes
(see
Table 2). B: I.itness of one In(2L)/,iSl genorype is similar to fitness of all In(21,)t and ST
homokaryotypes but low'er than the other three In(2L)t/ST genorypes. C: situation lbr no
heterokaryofypesuperiority; hetcrokaryotypesuperiority simrlar for all In(2L)t/ST genotypes;
heterokaryotype
superioritylor In(2L)t/SThcterozygousfor Ádh andfor aGpdh.respectively.
142
Summarizing
drscussron
Table 4. Theoretical fitness values of STi,9'l'.In(2L)t/hr(2L)t and the four In(2L)t/St genotypes
leading to equilibrium frequenciesfor Adh, a{}pdh and In(2L)t and gametic disequiltbrrumvalues
(D/D,,,,")betweenthe allozyme locr as observedin populationsfrom different geographicregions.
Simulations(1000 generations)were perforrnedby assun-ring
randommatrng,infinite populationsize
and no reconbination in In(2L)t/ST heterokaryotypes.
s/Í : ratio of selectioncoeÍïcients against
(seeFig. l).
In(2L)t (s) and Sl(/) hornokaryotypes
Fitness valuesIn(2L)t/ST
s/q'
sË
HF
Equilibrium frequencies
HH
s/t
Adh"
aGpdh" In(2Ll
D/D^",
Tropicalregions:
1.18
t.20
1.20
l.t8
1.20
1.20
1.15
t.2Q
1.20
1.15
t.20
t.20
Mediterraneanregions:
1.00
1.00
L00
1.00
1.00
1.00
1.00
1.00
1.00
1.10
1.00
1.10
1.00
1.00
1.00
1.00
0.90
0.87
0.84
0.80
0.90
0.87
0.84
0.80
0.50
0.50
0.50
0.50
0.01
0.01
0.01
0.09
LlO
1.10
1.10
1.10
1.10
1.10
1.10
1.10
3.00
4.00
5.00
6.00
0.25
0.20
0.17
0.t4
0.63
0.ó0
0.58
0.s7
0.25
0.20
0.r7
0.14
1.00
1.00
1.00
r.00
1.04
1.04
1.04
1.04
1.04
1.04
|.04
1.04
t2
16
20
24
0.18
0.22
0.27
0.30
0.54
0.53
0.52
0.51
0.08
0.05
0.04
0.03
0.38
0.20
0.13
0.09
Temperateregions:
1.00
1.00
1.00
1.00
1.00
1.00
1.00
r.00
The time neededto reach the equilibrium frequenciesdependson the level of
overdominance
andtakeson average250 generations
for the examplesin the tropicsand450
generationsfor the Mediterraneanclimates. After simulation for 1000 generations,
equilibriumfrcquencieswere not reachedin the caseof the fihressrelationspresentedfor
climates(basedon the lorv In(2L)t frequenciesin this climate),though for the
temperate
uppertwo examplestheAdh and aGpdh allelc frequencies
and the associationlevel between
AdhsandIn(2L)t werecomparablewith thosefrom naturalpopulations.This suggeststhat D.
populationsfrom temperateregions may not be in equilibriurn.An other
melanogaster
possibilityis that selectionintensityon the allozymepolymorphismsis higher in temperate
thanin íopical and Mediterranean
clintates
regions.This is supportetlby the strongereffects
of density(restrictedfood conditions)and ethanol (chapter 4) on the Adh and,dlpdh
polymorphisms
comparedto effectsof varioustemperatures
(chapter 3).
The resultspoint to a large effect of In(2L)t on the maintenanceof the geographic
distribution
and on the seasonalfluctuationsof the Ádh and oGpdh allele frequencies.
The
of this effecthighly dependson the fractionof Adhs/diptl/zrhaplotypesassociated
magnitude
143
wtÍh hr(2L)t and otr the fitnessrelationsbetweenlreterokaryotypes
r.vithdifferentAtth/ffipdh
haplotypeson the SZ chromosome.Moreover,the geneticcontentof In(2L)t chrontosomes
may vary among geographicregions,resultingin different interactionswith the allozyme
polymorphisms.
DNA variationin In(2L)t and ^SIchromosomearrangements
In(2L)t inversionsare expectedto have originatedfrom a unique rnutationalevent and to
possesslowcr levels of variation than SI chromosomesdue to the strongly reduced
recombitrationin In(2L)I/ST heterokaryotypes.
This hypothesiswas studied with three
different DNA-basedteclrniquesas thc resoivingpower of allozynre electrophoresis
was
insufficient (chapter 6). DNA variation in In(2L)t and ,SZ chromosomesfrom a Dutch
(Groningen)and a French(Vemet, PyreneesOrientales)populationwas surveyedat three
levels,with the following techniques:1) the whote h(2L)t region (about 13000 kb) was
assayedwith the Random Amplified PolymorphicDNA (RAPD) tcclurique,2) two gene
regions associated with In(2L)t !ilere analysed for Restriction Fragmcnt Length
Polymorphisms(RFLPs):Adh [closeto the proximalh(2L)t breakpoint]and d]pdh located
in thc centralparltof In(2L)tl, 3) part of rhe ocpdh gene(0.6 kb) including exon an<lintron
regionswas analysedby meansof DNA sequencing.
A summary of genetic diffcrentiationbetrveenIn(21)t and SI as revealedby the
differcntDNA techniques
is prcsentedin Table5.
Table5. Differentiation
withinandbetween
In(2L)tand,Slchromosomes
froma Dutchanda southem
French
D. ntelanogaster
populatron,
assessed
u'ithdilferentmolecular
techniques.
Technique
Within populations
Groningen
Vernet
Betweenpopulations
In(2L)t
Standard
A
RAPD
NFLP Adlt
RFIP aGpdh
sequencingffiptlh
+
-r
+
+
+
+
B
alloz)'mes
RAPD
+
A. Within thc In(2L)t legion (I{APD : whoL' In(2L1t region, about 13000 kb; ITFLP : the y'rl/r gene
regron [2.7 kb probe] close to the proximal In(2L)t breakpoint, and the aí3pdh gene region [6.0 kb
probe]locatedin the centralparto1'theinversion;sequencing: 0.6 kb of the dlpdh structuralgene).B.
Outsidc the In(2L)t region (allozymcsand RAPD : random samplingof the rest of the genome).
+:
significantdifferentiatron;- : no drftèrentiation.
Summarizingdiscussion
In addition, allozymeloci locatedoutsidethe In(2L)t region (1/:17; 11 polymorphic)and
randomlydistributedover the rest of the genome.showedno geographicdifferentiationwithin
andbehveenSI and h(2L)t. This indicatesthat for allozymesthe genomicbackgroundsfbr SI
andIn(2L)t arenoï differentiatedboth at the intra- andthe inter-populationlevel.
DNA variation in the whole In(2L)t region
Although the RAPD techniquedetectspolymorphismsthroughout the genome, it was
possiblewith specially designedExperimentalLines (ELs) to localise RAPD markers
rvitlr the h(2L)t region.The constructionof theseELs was performedin such a
associated
way that the genomicbackgroundsof In(2L)t and SIwere similar in eachof the ELs. Thus,
theELs arepolyrnorphicfor Íhe In(2L)t regiononly (i.e. about 15% of the gcnome),and SZ
andIn(2L)t homo- and heterokaryotlpesare presentin each of these lines. The three
karyotypescan be identified by different Adh/aGpdh genotypes:AdhssiaGltdh" double
homozygotesare homozygous for In(2L)t, double honozygotes Adht'/qcpdhss are
homozygousfor SZ and double heterozygotesfor Adh and aGpdh are heterozygousfor
ht(2L)tandSZ(Fig. 2). Only 15 out of 63 observedpolymorphicDNA fragmentsdetectedin
ÍheIn(2L)í region were unique for a karyotype,whereas26 were unique for a locality and22
wereabsentin one locality anduniquefor a karyotypein the otherlocality.Tlris indicatesthat
ht(2L)t harbours a surprisingly high level of RAPD polymorphisms. Geographic
In(2L)t and .5I for
differentiationfor In(2L)t, \Á,aseven higher than differentiation betr,r,een
eachof the populations.Moreover,polymorphismssharedby the two chromosometypes
wereobserved,
indicatingat leastsomegencticexchangebetweenIn(2L)t and,SZ.
Adh
1
z
3
4
Gndh
-r",,
,:'r$rr:i:,ql
,t,*
,r;:;1$:
I
:L$
,f,:$&.$,
,'g;
',,@r
:$
5
^
,:*l
,.::rr:!&ali
lg:.
.r:::t::t:t
t:
,*
7
8
:Wry
9
,,',,,,*.&'r,Êi
10
Figure 2. Ádh and djpdh
,*,
.*[w.
.
,*
s,
a|\ozyme banding patterns of individuals liom a specrally designed
line. Left zone of bands:Adh; right zone: diptlh lsee text). Lanes 1, 3, 6, 8 and 9:
expenmental
Adhsriacp(thsn
double hetcrozygotes(tn(2L.)t/Sl heterokaryotypes);lanes2, 4 and l0: ÁdhFts/(íipdhss
homozygotes(SZ homokaryoflpe); Lanes 5 and 7'. AdhstidlpdlFF
homokaryotypes).
-4L
liomozygotes (In(2L)t
Chapter7
DNA variutir,tnin the Adh and diptlh gene regions
DNA polymorphisms,detectedwith the RÁPD technique,are locatedthroughoutthe whole
In(2L)t region. Analysesof the Adh region (closeto the proximal In(2L)t breakpointand
reflectingthe evolutionaryhistory of the inversion)and Ílte a{}pdh region (locatedin the
celltral paÍ of the inversion)supply infomralion concernin,q
levels of DNA variation and
differentiationwithin andbetweenIn(2L)t and5Z in differentpartsof the In(2L)t region.
DNA variation in the Adh region was measuredby the use of RFLP analysis.
Nucleotideheterozygosityfor In(2L)t and ,SZwasestimatedas 2.01 * 10-rand8.20 * 10-3,
respectively.Thus, two randornlychosenIn(JL)t chromosomesdiffer for one out of 500
nucleotides,
while two 5'I chromosomes
diffcr by one out of 122 nucleotidesin the Adh
region.A higherlcvel of nucleotidevariationlor Slcompared ïo In(2L)Í hasbeenobserled
for both populations,while geographicdiffercntiationwas absentfor eachof the chromosome
alTangements.
RFLP as well as sequence
dataof the aGpdhregionshovveda low level of variationfor
In(2L)t from the Groningenpopulation.s,hile the level of variation for In(2L)t from the
Vernet population was similar to SL The latter arrangementshowed similar levels of
nucleotidevariationfor within and bctweenpopulationcomparisons.Polymorphismsshared
by the two chromosome types were found in the Vernet population. Nucleotide
lreterozygosiÍy
in the aGptihregionwas estinrated
as 12.ó3*10'rfor In(2L)rand as 7.50*10-r
for SL Thus,the overall(populationscombined)level of DNA variatiorrin Íhe ffipdh regíon
is evenhigher for In(21,)tÍhan for ST.
In(2L)t showeda six-fold higherlevel of DNA variationfor the aGpdh regionthanfor
Adh
rcgion(two-tailedWilcoxonsignedrank test;P < 0.01),while .lI showedsimilar
\he
ievelsof DNA variationfor tlie two generegions.Geneticexchangcivithin the In(2L)t region,
e i t h e r b e t w e e n ï w o I n ( 2 Lr e) tg i o n s o r b e t w e eI nn( 2 L ) ta n d S Z b y m e a n s o f t h e o c c u r r e n c c o f
doublecrossoversand/orgeneconversion(Rozasand Aguadé I 994; Popadicet al. 1995),is
expectedto happen more frequently with increasingdistance from the chromosonral
breakpoints(Ashbumer 1989).Consequcntly.it will occur more frequenÍlyin lhe aGpdlt
regionthanin theAdh region.The discrepancy
for theAdh andaGpdh regionswith respectto
levelsof variationamongIn(2L)t and SZ arrangements
may thereforebc explainedby their
chromosomalpositions in relation Ío Íhe In(2L)l breakpoints,rather than by different
selectionpressurcs
actingon ÍlteAdh andaGpdhpolymorphisms.
Geographicvariation ín In(2L)t content
As inversionfrequenciesare generallyvery low in temperateclimates,but much higherin
Mediterranean
climatesand tropicalregions,geneticexchangeamongIn(JL)t chromosomes
and betweenltt(2L)t and SI chromosomes
will occurmore frequentlyin the southcmFrench
populationthan in the Groningenpopulation.This agreeswith the relatively high levelof
DNA variation in the aGpdh region for In(2L)t lrom Vcrnet, the presenceof shared
polymorphismsandthe absenceof differentiationbetweenIn(2L)t and.SI in this population.
The different levels of DNA variationin the aGpdh region for In(2L)t from the two
populationsand the distinctionin differentiationbetweenIn(2L)t and Sl may'be ascribed
to
stochasticprocesses
likc foundereffectsand/orbottlenecksin effcctivc numberof inversion
140
l
Summarizingdiscussion
karyotypes.In this respcct,observedDNA variation in the aGpdh region associatedu,ith
In(2L)t, is positively correlatedwilh In(2L)t frequenciesin the populations.In addition.
In(2L)tderivedflom thc Groningenpopulation,containssix uniquenucleotidepositionsin a
605 bp aGpdh region. This can partly bc explainedby the maintenanceof spontaneous
recessive mutations (even those which aÍe deleterious) in heterokaryotypesby
heterokaryotype
superiority(chapter 3, 4 and 5), leadingto fixation in the inversionby
chancc.Moreover, lel'els of genetic variation within and between In(2L)t and SI
anangements
from differentgeographicareasprobablydependon the historicaldistribution
patternof the species(Veuille et al. 1998).
The resultsindicatethat the geneticcontentof In(21.)tvariesamonggeographicregions
andthereforemay vary with latitude.The latitudinal distribution rn In(2L)t lrequenciesmay
thenpartlybe causedby geneticcontentco-varyingr.r'ithlatitude,e.g.as obsened for insertiondeletiorrpolymorphismin the Atlh gene(Berry and Kreitman 1993).Consequently,
fitness
differencesbetween individuals carrying In(2L)t and/or ,SZ chromosomesmay vary with
geographic
origin. Fitnesscomponentsor fitness-related
characters
such as high-temperature
rveight
which
resistance,
developmentalrate and body
show consistentfitnessdifferences
betweenkaryotypesfrom different geographicorigins (Van Delden and Kamping 1991, 1997
Van 't Land i997; Kamping and Van Delden 1999b),are expectcdto be (more frequently)
controlled
by geneslocatedin breakpointregions.
Synopsis
ol DNA variatiortin Irt(2L)tantl ST
Althoughcy4ologically
identicalnaturalinversionsmay not ahvaysbe monophyleticin origin
(Caccone
et al. 1998),theobservedresultsdo not contÍavene
a uniqueonginof In(2L)t: theAdh
region(closeto the proxintalbreakpoint,reflectingthe evolutionaryhistory)showsa four-fold
lorverlevel of nucleotidehetcrozygosityfor In(2L)t than for SI and a low level of genetic
exchange
betweenIn(2L)t and SZ.In view of the lack of differentiationwithin In(2L)/ aswell as
between
In(2L)r and SI in this region,thc invcrsionht()1,)tprobablyhas a relativelyrccent
origin.
In(2L)t chromosomesexhibit a considerablclevcl of DNA variation and geographic
differentiation
asdctectedwith RAPD analysisof thewholeIn(2L)t regionandDNA analysisof
thedipdh region.The differencein levelsof geneticvariationbetweenIn(2L)t andSZ depends
positionandnatureof the DNA stretches
onthegeographic
origin but alsoon the chromosomal
The higher level of differentiationobtainedwith the RAPD technique
underinvestigation.
compared
to RFLP and sequencing
of two functionalgenescan be explainedby "sampling"
differcnttlpes of DNA. RAPD fragmentsmainly consistof non-functional(neutral)DNA
while the two allozymegenescontainfunctionaland selecti'"'cly
sequences,
constrained
DNA
with a low rateofevolution.
sequences
Despitcthe substantialgeneticexchangcbetn'eenh(2L)t and SZ ín thc aGptlh region,
with the aCpdhFallele.The low numberof silentnuclcotide
ln(2L)tís exclusively
associated
polynrorphisms
observed around the allozyme determining site of aGpdh rn In(2L)t
can be explainedby directionalselectionon that site (favouringthe F allele)
chromosomcs
leadingto reductionof nucleotidevariationat nelrtrallinlied sitesby hitchhiking(Aguadéel
c/. 1989;Charlesworthet al. 1993). Thus the pattern of polymorphic nucleotidesites
1Àa
-.4.-
Chapter 7
indicatesthat the association
betweenIn(2L)t ar'rcL
aGptlht is maintainedby naturalselection.
Particularallelic combinationsin the In(2L)l region which are of adaptive significance
apparentlyaremainlainedby purifying selection.
Concludingremarks
The study of three genetic polymorphismssirnultaneouslyunder highly variable natural
conditionsand variousgeneticallyand environmentallycontrolledexperimentalconditions,
enablesthe analysisof interactionsbelweenthesepolymorphisms.The resultspoint to very
complex interactiotlsin natural D. melanogctslerpopulations,dependingon allele and
karyotypefrequencies,
on environmcntalconditionsand on populationdemography.Studies
on geneticvariation of a single geneticpolymorphismcan thereforeeasily lead to wrong
conclusionsabout the mechanismsunderlying the maintenanceand evolution of that
polymorphism.By ignoringIn(2L)t polymorphisme.g.effectsof naturalselectiononthe Adh
(mostly overestimated),as h(2l)t is
andlor acptlh polymorphismcan be rrrisinterpreted
with the ÁlhslacpdhL allelecombination.It is clearthat hitchhikingeffects
alwaysassociated
of the allozyme polymorphismswill increasewith increasingfractions of AdhslacpdhF
haplotypes associated with In(2L)t. In natural D. melanogasÍer populations from
wilh In(2L)t (e.g.Van 't Land
Mediterranean
climates,(alnrost)all Adhsallelesareassociated
et al. 2000). Frequency fluctuations at lhe Adh iocus in those climates can therefore be
explainedby selectionon the In(2L)r polymorphismwith passivehitchhiking of the Adh
polymorphism.This probablyexplainsthe instability in Adhs frequenciesin Mediterranean
climatesas describedby David et al. (1989).
The large fitness differencesbetween h(2L)t and SZ karyotypes (a gene region
containingabout2000 genes)which are observedunderparticularenvironmentalconditions
can be furtherexploredby identifyingthe genesthat affect fitness.The observedkaryotype
diagnosticDNA markerscan be usedas a startingpoint for the localisationand identification
of genes.Sincethe completionof the D. melanogastergenomeproject in March 2000,the
DNA sequences
and chromosomallocationsof all genesin ïhe h(2L)l region are known.
This infonnationcan be usedfor measuringdifferencesin geneexpression,if any, between
In(2L)t and SZ karyotypesunder particular environmentalconditions. The micro-array
procedureallows measurements
of exprcssionof many genessimultaneouslyundervarious
experimentalconditions.This molecularapproachlooks promising in detectinggeneswith
different levels of expressionbetweenIn(2L)t and SI karyotypes.Both populationgenetic
and moleculargeneticresearchwill be necessary
to link thesedatawith fitnessdifferences
at
the individualas well as at the populationlevel.It will be a big challengefor futureresearch
to characterise
and localisethat part of the 2000 genesin lhe h(2L)t region which affects
fitnessand to investigatehow epistaticinteractionsand/orpleiotropiceffectsbetweenthose
genescontributeto fitnessdifferencesbefweenkaryotypes.
148