doi 10.1098/rspb.2000.1235 Evolutionarydynamicsof decliningmelanism inthepepperedmothinTheNetherlands Paul M.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 similarity betweenthe 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 other areasof 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 other regionsof 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, Proc. R.Soc.Lond. B (2000) 267, 1953^1957 1953 © 2000The RoyalSociety Received 9 May 2000 Accepted 22 June 2000 1954P .M. Brake¢eld and T.G. Liebert Declining melanism inDutchpeppered moths 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