Evolutionary Dynamics of Declining Melanism in the Peppered Moth In

doi 10.1098/rspb.2000.1235

Evolutionarydynamicsof decliningmelanism inthepepperedmothinTheNetherlands PaulM. Brake¢eld 1* and Tony G.Liebert 2 1Institute of Ecological and Evolutionary Sciences, Leiden University,POBox 9516, 2300RALeiden,The Netherlands 2`The Heath¢eld’ ,Crowcombe Heath¢eld, NearTaunton, Somerset TA44BT,UK Populationsof Biston betularia inthe regionof The N etherlands aroundLeiden and Rotterdam were resampled. Acomparisonof three sets ofdata for 1 969^1973,1988 and 1999 enabled a further examinationof declines inmelanism. Unlikeparallel changes for the black carbonaria formof this species inurban regions of Britain, those inThe Netherlands involvesubstantial changes in frequencies ofatleast twoof the intermediate insularia morphs aswell as an increase inthe non-melanic typica morph. The darkestof the three insularia morphs hasshown a transitorypulse ofincreased frequency in TheN etherlands.The dynamics are discussed inrelation to the historyof air pollution and to straightforwardpredictions about selection. Keywords: industrialmelanism ; Biston betularia ; carbonaria morph;natural selection ;adaptation;evolution

haveindicated some similaritybetween the dynamicsof 1.INTRODUCTION the declineof carbonaria innorthern England and that of Theevolution of industrial melanism inthe peppered swettaria inMichigan, USA (Grant et al.1996,1 998;Cook moth (Biston betularia )hasbeen thoroughly documented in et al. 1999). Britain.Steward ( 1977)was able to use acombinationof Asin Britain, in the mid-20thcentury the central, the literature andmuseum collectionsin tracking the urbanregions of The N etherlandswere associated with earlierspread of the black carbonaria morphfrom its melanism innumerous species ofmoth,high air pollution initialsightings in the mid-19thcentury .Thisfollowed andan absence of epiphytic lichen growth on trees (see Haldane’s(1924)well-known calculation of the ¢tness Barkman1 969;Kettlewell 1973).Thepresent paperfollows advantagenecessary foraccounting for the spreadof upan earlier report (Brake¢eld 1 990)on B.betularia inThe carbonaria.Rich datasets collectedby other researchers Netherlands,which indicated a sharpdecline in carbonaria havedocumented the spatialcorrelation between melanic fromaround 1 970until 1 988.Bakker et al.(1987)found an frequencies andlevels of air pollutants during the mid- increase inspecies richness ofepiphytic lichens onfour 20thcentury when allele frequencies appearedto be more species oftree, includinglimes ( Tilia spp.),inthe same orless stable (see the reviewsby Kettlewell 1973;Lees regionbetween 1 973and 1 984.This was correlated with a 1981;Majerus 1998;Cook 2000) .Kettlewell (1955,1 973), progressivedecline in sulphur dioxide that hadbegun Bishop( 1972)and Bishop et al.(1978)obtained estimates approximatelyten yearsearlier .Bythe mid-1980sthis ofthe relative¢ tnesses ofmelanic and non-melanic componentof air pollution had declined by ca. 90% (see morphs andof migration rates. Inaddition, several ¢g.3 inBrake¢ eld ( 1990)).Weresampled severalpopula- surveysin more recent decadeshave documented tions of B. betularia in1999 in order to document whether dramaticdeclines inthe frequencyof the carbonaria the declinein melanism hadcontinued and to examine morph,which are associated with decreasing air pollution further howfrequency changes in carbonaria have been andchanges in relative ¢ tness. Themost complete data accompaniedby increases inother morphs. The earlier set is that collectedby Sir CyrilClarke for Caldy in report hadsuggested more involvementof insularia morphs north-west England(Clarke et al.1985,1994; also see thanin northern England. These intermediate melanic Majerus 1998;Cook et al.1999).Thedecline in carbonaria morphs arecontrolled by three additionalalleles at the throughoutmuch ofurban Britain has been largely carbonaria gene( Lees &Creed1 977).The insularia morphs matchedby an increase inthe wild-type,non-melanic haveapparently remained uncommon during the recent typica morph.Three sets ofdata collected in England decline of carbonaria innorthern England (e.g. Cook et al. providedestimates ofthe ¢tness of carbonaria during this 1999).Theymay have been historically more abundantin periodof declining melanism asbeing 79^89% that of otherareas of Britain and northern Europe and insularia typicals(Cook et al.1986,1 999). mayplay a more prominentrole in regionswhere levels of Industrialmelanism hasalso been described inpopula- airpollution have changed more graduallyor less mark- tions of B. betularia fromsome otherregions of Europe edly(see Kettlewell 1973;Lees 1981;Majerus 1998). (see Lees 1981)andin the NorthAmerican subspecies Biston betularia cognataria (melanicmorph swettaria) (see 2.MATERIAL AND METHODS Majerus 1998).Itistherefore ofgreat interest tocompare anyparallel changes in melanism insuch populationsto Extensivecollections of B.betularia from1 969to 1 973from those occurringin Britain. Comparisons of recent throughoutThe Netherlandswere produced using light traps samples of B.b.cognataria withthose fromaround 1960 underthe coordination of B. J.Lempke (as reported by Kettlewell1973) .Analysisof this material, largely using *Author forcorrespondence (brake¢ [email protected] .nl). Lempke’sdata, was described by Brake¢ eld (1 990).Inaddition,

Table 1. Numbers of the ¢vemajor non-melanic and melanic morphsof thepeppered moth B.betularia insamples obtained from the indicated localities in1999 (I1^I3are the three intermediate insularia morphsfrom pale to dark. Each sample is divided into portions collected early and laterin the£ ightseason.)

numbers localityand sample typica 11 12 13 carbonaria total

Leiden early 34 4 11 7 1 57 late 19 5 8 3 0 35 total 53 9 19 10 1 92 Voorschoten early 33 8 8 9 0 58 late 127 20 30 27 2 216 total 160 28 38 36 2 264 Schiedam: early 11 2 8 6 0 27 late 27 5 8 14 4 58 total 38 7 16 20 4 85

(a) (b) (c) Inthe present study ,we resampledpopulations in Leiden, 60 n = 92 n = 264 n = 85 nearDen Haag (at V oorschoten)and near Rotterdam (at Schiedam)in June1999. The localitieslie approximately along a 40 northto south transect of ca.30kmin lengthwith Voorschoten lessthan 10 km south of Leiden. Assembly traps baited with 20 rearedfemale moths were again used for collecting adult males. The trappingsites at V oorschotenwere near or atthe edge of a e

g 0

a largearea of woodland, while thoseat the other two localities t

n (d) (e) (f ) e werein moreurban environments. c

r 60 e n = 340 n = 195 n = 258 Mothswere scored as in earlierstudies ( Brake¢eld 1 990).They p wereclassi¢ ed into ¢ vemorphs: black carbonaria, three 40 intermediate insularia (I3,I2 and I 1fromdarkest to palest) and non-melanic typica.Thesemorph classes follow the dominance 20 hierarchyfor the ¢ vealleles, with carbonaria thetop dominant and 0 typica thebottom recessive ( Kettlewell1973; Lees & Creed1977) . Leiden Voorschoten Schiedam Asin theearlier survey ,mistakesin classi¢cation were most likely (g) tooccur at the boundaries between carbonaria and I3 and betweenI 1and typica.However,most moths were in goodcondi- 60 n = 232 tionwhen inspectedand the key characteristics involving upper e g

a hindwingpatterning and dorsal body coloration usually provided t 40 n

e littleor no uncertainty ,evenat these boundaries (see Brake¢ eld c r

e 1990).The mid- insularia (I2)is particularlydistinctive.

p 20 Zuid-Holland

0 tt C/? 3. RESULTS

Figure1. Changes in morphfrequencies in B.betularia at Table1 givesthe morphfrequencies for1999 from the localitiesin theprovince of Zuid-Hollandin The Netherlands localitiesin Zuid-Holland and the datafor all three overthe three collecting years: ( a^c) 1999, (d^f ) 1988 and surveysare shown graphically in ¢ gure1. Carbonaria (g)1969.The increaseddegree of shadingfrom left to right declinedfrom ca.70%in 1 969^1973to ca. 10% in 1988 representsthe ¢ vemorphsin sequence: typica (tt), insularia 1, (witha lowerfrequency at Schiedam near Rotterdam) . insularia 2, insularia 3 and carbonaria (C/?). Thedecline appears to have continued up to the present day,withonly seven out of 44 1moths collectedin 1 999 severallarge samples of the moth were collected using assembly beingscored as carbonaria.Table2 givesthe pooled trapsin 1988in theprovince of Zuid-Holland near the cities of samples forthe three surveysin Zuid-Holland and Leiden,Den Haag (at V oorschoten)and Rotterdam (at includesestimates forallele and morph frequencies Schiedamand Hoogvliet) .Thesesamples were compared with assumingHardy ^Weinbergequilibrium (see Hartl & thepooled data from Lempke’ ssurveyfor all collecting sites Clark1 997,pp.88^92) .Thereis some correspondence within thesame province (which hadyielded homogeneous betweenthe estimates ofthe allelefrequencies andthe samples).The carbonaria morphfrequency had declined from ca. observedmorph frequencies. Inparticular, the increased 70to 10% over 16^1 9generations(Brake¢ eld 1 990). frequencyof the darkest insularia morphin 1988 was

Proc. R.Soc.Lond. B (2000) Declining melanism inDutchpeppered moths P.M. Brake¢eld and T.G. Liebert 1955

Table 2. Numbers and morphfrequencies for pooled samples of the peppered moth B.betularia from the Dutch province of Zuid-Holland in each sampling period together with estimates of allele and morphfrequencies assuming Hardy ^Weinberg proportions sample typica 11 12 13 carbonaria total numbers 1969^1973 10 18 23 18 164 232 1988 331 70 77 259 56 793 1999 251 44 73 66 7 441 allelefrequencies 1969^1973 0.208 0.139 0.122 0.072 0.459 ö 1988 0.646 0.065 0.065 0.188 0.036 ö 1999 0.754 0.064 0.095 0.079 0.008 ö observedmorph frequencies 1969^1973 0.043 0.078 0.099 0.073 0.707 ö 1988 0.417 0.088 0.097 0.327 0.071 ö 1999 0.569 0.100 0.166 0.150 0.016 ö expectedmorph frequencies 1969^1973 0.043 0.077 0.100 0.073 0.708 ö 1988 0.417 0.088 0.096 0.326 0.072 ö 1999 0.569 0.101 0.164 0.150 0.016 ö accompaniedby a higherallele frequency .Thefrequency Netherlands.The dominant carbonaria allelehas shown a of the carbonaria alleledeclined over the 30generationsof sharpdecline in frequency .Therate ofdecline is ofa the wholesampling period from ca.0.5to less than0.01, similar orderto that observedin northern England whilethe bottomrecessive typica alleleincreased from (Cook et al.1999).However,in comparison with the latter ca.0.2to 0. 75. populations,where the carbonaria alleleis beingreplaced Theearlier periodof the declinein carbonaria was by the typica allele,there is anindication of a more matchedby increases inthe darkest insularia (I3) and in complexdynamics involving changes in morphs speci¢ed the non-melanic typica.Theincreases wereclear for both byother alleles at the carbonaria locusin The N etherlands. morphand allele frequencies (table2) .Therewas little if Thefrequency of the non-melanic typica hasincreased anyresponse inthe other insularia morphs (I2and I 1), progressivelyfrom around 1 970to the endof the century. althoughthe allelefrequencies mayhave declined. In Initially,there wasalso a markedincrease inthe darkest contrast, there wasan increase inthe mid- insularia (I2) insularia (I3).Thisphenotype of insularia then declined morphof up to ca.15^20%during the last decade,which withsome reductionin allele frequency ,whilethe paler appearsto have been accompanied by a small increase in mid- insularia (I2) morphbecame more abundant, allelefrequency (table 2) .Theincrease in typica also althoughwithout such asubstantialincrease inallele continued.These changes occurred largely at the expense frequency(see table2) . ofthe darkest insularia (I3).Theseobservations of pheno- Thus,the declinein carbonaria inurban environments typicchange were supported by heterogeneity w2-tests ofTheN etherlands isbeingaccompanied not only by an performed across years(with or without combining increase in typica non-melanics,but also by changes in carbonaria andI3) ,althoughcomparisons involving insularia intermediates. Atleast oneof the latter changes Schiedamonly approached signi¢ cance (see alsoBrake- hasinvolved a periodof increased abundance, which was ¢eld1 990).Thetemporal changes in morph frequencies apparentlyaccompanied by a rather sharpincrease in werebroadly or closelyparallel across localities. allelefrequency .Grant et al.(1996)found that the Themoths weretrapped over the whole£ ightperiod frequencyof the class of insularia morphs increased from1 1Juneto 1 6Julyin 1 999.A markedpeak in slightlyat Caldy in north-west Englandover a periodof captures occurredin the secondweek of J uly,particularly monitoringthe declinein the carbonaria morphfrom 1 959 atV oorschoten.Dividing the captures intoan earlyand a to1993. Our examinationof the same extensivedata set lateperiod ( 11June^1Julyand 2^16 J uly)enabled exam- forthe Caldylocality ( Clarke et al.1994)showed that the inationof any change in morph frequencies withinthe transitionthere from carbonaria to typica hasoccurred season(table 1 ).Notrends wereapparent ( w2-test statistics withoutany other changes in allele frequencies (¢gure 2) . alwaysnon-signi¢ cant whether using four classes withI3 Thus,assuming a single insularia alleleof intermediate and carbonaria combined or typica versus allmelanics dominance(the insularia morphs werepooled) and combined). Hardy^Weinbergproportions, the allelefrequency remainedremarkably constant over this periodat around 0.02(only during the periodof very rapid decline in the 4.DISCUSSION carbonaria morphfrom 1 976to 1986 were frequencies in Carbonaria hasbeen transformed fromthe most severalalternating years a little higher).Incontrast, over commonto the rarest morphin populations in less than aperiodof 30 yearsin The N etherlands, insularia morphs 30generations in central, urban regions of The haveshown an overall increase infrequency whilst the

Proc. R.Soc.Lond. B (2000) 1956P .M. Brake¢eld and T .G. Liebert Declining melanism in Dutchpeppered moths

0.07 straightforwardexplanation may be involved since, ifthe insularia morphs havea sequence ofintermediate relative

y 0.06 ¢tnesses, successive rises andfalls would be expected in c n

e their frequencies evenif their ¢tness valueswere constant

u 0.05 q

e intime duringthe decline(L. M. Cook,personal r f 0.04

e communicationafter examinationof simulation data) . l e l

l Sucha scenariodoes not necessarily involve any

a 0.03

a complexdynamics, for example in terms ofsuccessive i r

a shifts inselection gradients.Whatever the precise details l 0.02 u

s ofthe biologicalreasons why spatial variation and n i 0.01 temporalchange in relative ¢ tness occurin B. betularia , 0.00 the documenteddeclines inmelanism providefascinating examplesof dynamicevolution. 1960 1970 1980 1990 year Wededicatethis paper to Sir Cyril Clarke FRS who contributed substantiallyto the understanding of industrial melanism and Figure2. Estimates of thecombined frequency of the insularia whodid much to stimulate our own interests in theLepidoptera. allelesfor the Caldy population of B.betularia obtainedfrom Wearevery grateful to Dr L. M. Cookand Dr B. Grantfor analysisof themorph frequencies given by Clarke et al. (1994) theirmost constructive comments on the manuscript. W ealso andunder the assumption of Hardy ^Weinbergproportions. thankM. Brittijnfor preparing the ¢ gures. The averagesample size per year was 494 moths. pooledallele frequency has declined by approximately 0. 1 REFERENCES (see table2) . Regularvisual inspections oftrunks oftrees growingin Bakker,A. J.,V anDobben,H. F.,TerKeurs,W .J.&Meelis,E. Leidenand its environsover the last decadehave shown 1987T erugkeervan epifytische korstmossen in Zuid-Holland. that the earlierincrease inspecies richness ¢rst docu- Landschap 1987, 4^18. Barkman,J. J. 1969 The in£uence of airpollution on bryophytes et al mented byBakker .(1987)has now yielded dramatic andlichens. In Airp ollution ,pp.197^20 9.W ageningen,The changesin the coverageof tree trunks andupper canopy Netherlands:PUDOC,Landbouw U niversiteitW ageningen. branchesby crustose andfoliose lichens (forfurther Bishop,J .A.1972An experimentalstudy of the cline of discussion see Liebert &Brake¢eld ( 1987)).Forexample, industrialmelanism in Bistonbetularia (L.) (Lepidoptera ) the south-west sides ofthe trunks ofextensive 30^50- betweenurban Liverpool and rural North W ales. J. 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Proc. R.Soc.Lond. B (2000) Declining melanism in Dutchpeppered moths P.M. Brake¢eld and T.G. Liebert 1957

Kettlewell,H. B. D.1 973 Theevolution of melanism .Oxford,UK: Liebert,T .G.& Brake¢eld, P .M. 1987Behavioural studies on ClarendonPress. thepeppered moth Bistonbetularia anda discussionof therole Lees,D .R.1981Industrial melanism: genetic adaptation of ofpollution and lichens in industrialmelanism. Biol.J .Linn. animalsto air pollution. In Geneticconsequences of man made Soc. 31, 129^150. change (ed.J .A.Bishop& L.M. Cook),pp.1 29^176.London: Majerus,M. E.N.1 998 Melanism:evolution in action . Oxford AcademicPress. UniversityPress. Lees,D. R. &Creed,E. R. 1977The geneticsof the insularia Steward,R. C. 1977Industrial and non-industrial melanism formsof the peppered moth, Bistonbetularia . Heredity 39, in thepeppered moth Bistonbetularia (L.). Ecol.Entomol . 2, 67^73. 231^243.

Proc. R.Soc.Lond. B (2000)