Table Of ContentJournaloftheLepidopterists'Society
53(3),1999,99-103
AN EXAMINATION OF INTRASEASONALVARIATION IN THE INCIDENCE OF MELANISM IN
PEPPERED MOTHS, BISTONBETULARIA (GEOMETRIDAE)
Bruce S. Grant
DepartmentofBiology,CollegeofWilliamandMary,P.O. Box8795,Williamsburg,Virginia23187-8795,USA
AND
CyrilA. Clarke
EmeritusProfessorofMedicine,UniversityofLiverpool, LiverpoolL693BX,UK
ABSTRACT. Weexamineddailycatchrecordsofpepperedmoths(Bistonbetularia)takenoveraforty-yearperiodtodeterminewhether
melanicandpaleformsofthisspeciesflyatthesameordifferenttimeswithinseasons.Wealsocomparedtheemergenceratesofthetwoforms
fromrearedbroodstodeterminewhetherdifferencesindevelopmentalratesmightcontributetointraseasonalflightpatterns.Althoughmelanic
phenotypesdevelopslighdyfasterthandieirtypicalsiblingsinsomelaboratorybroods,thefieldrecordsshownoconsistentpatternforonephe-
notypebeingmorecommonthantheotherearlyorlateinthesamesummer. Theworkisdiscussedinthecontextofindustrial melanism for
whichthisspeciesisdieclassicexample.
Additionalkeywords: cognataria,f.carhonaria,industrialmelanism,f.swettaria.
Industrial melanism is a familiar textbook example From his observations oflarval developmental rates,
ofobservable evolution brought about by natural se- he proposed that pale peppered moths, as fast devel-
lection. Thetermpertains toincreases inthe frequen- opers, avoided pollution, and/or that the melanics, as
cies of genetically determined melanic versus pale slowdevelopers, "maybecapableofgettingridoftoxic
phenotypes in populations living in habitats modified substances." He cited no reference to support his
by regional industrial development and urbanization. statementthat"slowfeedingand acapacityforexcret-
The phenomenon has beenwell documentedin many ing noxious materials has been demonstrated out-
. . .
speciesofLepidoptera; however, attentionhas focused sidethe Lepidoptera" (Kettlewell 1973:85).
primarilyon the peppered moth, Biston betularia (L) Unfortunately, Kettlewells developmental analysis
(Geometridae). The broad aspects ofthe subjecthave ofpeppered moths was limited to one brood thatwas
been reviewed recentlyby Majerus (1998), Sargent et partially consumed by mice, and a second brood that
al. (1998), and Grant (1999). provided "no corroboration ofthe earlier results." He
The corpus ofexperimental work to date is consis- also acknowledgedthat he couldprovide no evidence
tentwiththeinterpretationthatselectivepredationon from samples of wild populations that industrial
the moths by birds is the primary, though not exclu- melanic forms change in frequency during the flight
sive, force driving the changes in the frequencies of periodwithin single seasons. However, he did discuss
peppered moth phenotypes (Majerus 1998). Ket- intraseasonal changes in phenotype frequencies for
tlewell (1955, 1956) provided the first quantitative ev- several other moth species which showwhathe called
idencethatbirds eatthe differentcolorphases ofpep- "ancient" (=stable polymorphism) melanism. For ex-
pered moths according to their conspicuousness on ample, paleAmathes glareosa increase late in the sea-
differentbackgrounds. His mark-release-recaptureex- son (the melanics appear early on); whereas, Cleora
periments also demonstratedthat the melanic pheno- repandata melanics increase, relative topale forms, as
types fared better than the pale forms in soot-black- the seasonprogresses.
enedwoodlands; whereas, the pale forms faredbetter In analyses ofseasonal catch records ofother moth
thanthe melanics inunpollutedwoodlands. species polymorphic for melanic forms, Bishop et al.
Kettlewell (1973) entertainedotherpossibilities be- (1978) concluded that melanic Gonodontis bidentata
side selective predation that might contribute, at least emergedlaterthanpale forms, and Sargent (1983) re-
in part, to the high incidence of melanism in moth ported slightincreases in melanism duringthe second
populations living in the vicinities ofBritish industrial halfofseasonsinPhigaliatitea. Asonlyfielddatawere
centers. He speculated that larvae developing in the available fromthese studies, cleardistinctionsbetween
early part ofthe summer feed on leaves that are less selection on the adults and developmental differences
contaminated by industrial pollutants than are the inemergence schedulesbetweenthephenotypeswere
older leaves that larvae feed on later in the summer. not possible. Equally problematical, S. Poitout (cited
100 Journalofthe Lepidopterists' Society
Table1. June(=A)andJuly(=B)catchrecordsofB.betulariaphenotypes(Mel. =melanics,T+I=typicalsandf.insularia)atCaldyCom-
monbetween1959through 1998.ThephenotypicdistributionsduringJuneandJulyofeachyeararecomparedbyG-testsofindependence.
Year Mel 1+1 c Year Mel T+I c Year Mel Ttl c Year Mel T+I c
59A 174 15 69A 349 25 79A 387 111 89A 52 114
59B 90 4 1.46 69B 216 20 0.67 79B 111 23 1.72 89B 13 40 0.91
60A 178 11 — 70A 658 68 80A 379 115 90A 36 68
60B 34 2 70B 112 10 0.18 80B 103 40 1.30 90B 15 35 0.33
61A 161 18 71A 109 8 81A 118 46 91A 102 337
61B 240 12 4.45* 71B 79 11 1.75 81B 178 65 0.08 91B 139 355 2.93
62A 159 17 72A 45 3 82A 43 15 92A 227 750
62B 588 38 2.56 72B 202 26 1.26 82B 45 19 0.22 92B 32 111 0.05
63A 725 67 73A 102 14 83A 219 111 93A 41 146
63B 185 18 0.03 73B 181 21 0.21 83B 226 133 0.88 93B 22 65 0.37
64A 238 25 74A 99 14 84A 172 105 94A 39 196
64B 335 37 0.03 74B 178 22 0.14 84B 43 32 0.56 94B 26 87 2.01
65A 361 40 75A 78 16 85A 189 144 95A 33 149
65B 70 7 0.06 75B 155 20 1.60 85B 271 256 2.34 95B 13 66 0.11
66A 163 18 76A 129 29 86A 213 198 96A 5 78
66B 110 8 0.93 76B 242 46 0.05 86B 169 225 6.78** 96B 12 106 1.12
67A 123 10 77A 68 12 87A 141 201 97A 12 165 —
67B 243 21 0.02 77B 360 50 0.46 87B 41 61 0.03 97B 5 56
68A 209 26 78A 91 28 88A 44 66 98A 14 113
68B 216 26 0.01 78B 205 31 5.92* 88B 29 40 0.07 98B 12 87 0.07
*p<0.05
**p<0.01
by Bishop et al. 1978) showed in laboratory strains of West Kirby England over aperiodof40years, begin-
Spodoptera exigua that melanic genotypes had a ningin 1959. The originalpurpose ofthe studywas to
longertotaldevelopmentthannon-melanicgenotypes; assess frequencychanges inthe melanic, pale, andin-
thus melanicindividualstendedtoemergefrompupae termediate phenotypes overthe course ofyears; these
laterthanthe non-melanics, butno fielddatawerere- annual changes have been reported elsewhere (see
ported. Clarke et al. 1985, 1994, Grant et al. 1996, 1998). To
The abovestudiesservetoillustratethatgeneralities determine ifthe different phenotypes fly at different
are not obvious regarding developmental differences timesduringthesame season,wereexaminedthedaily
and/orin intraseasonalvariations in flightpatterns be- catch records for each of the 40 years moths were
tween melanic and pale forms of polymorphic moth trappedatthis location.
species. Therefore,wehaveanalyzedtheflightpatterns Each season a mercury vapor (MV) light trap was
in B. betularia directly and comparedthe emergence operated nightly from 1 June through 31 July. When
rates ofmelanic andpale phenotypes. To determine if virgin females were available, an assembling (phero-
melanic and pale phenotypes fly at different times mone) trap was also used. No difference in the pro-
during the summer, we examined the daily catch portions of the phenotypes caught by one trapping
records ofpeppered moths taken at a single location methodortheotherhaseverbeenobserved(Clarkeet
over a forty-year period. To assess potential differ- al. 1994), butthe incidentaluse ofthe assemblingtrap
ences in emergence schedules, we recorded the eclo- does increase the total catch size. For our analysis,
sion sequences ofmelanic and pale phenotypes from therefore, we subdivided each season into early and
three largebroodsproducedfrom controlledcrosses. late halves based on calendar date (June versus July)
rather than by the mid-point (median) of the total
Materials and Methods numbers ofthe moths caughtwithin seasons. Wethen
Field studies of intraseasonal flight patterns. tabulatedthenumbers ofeachphenotype caughtdur-
Biston betularia were collected near CaldyCommon, ingthefirstandsecondhalvesofeachseason. Thenull
Volume 53, Number 3 101
The year-to-year sample sizes varied widely, ranging
between 122 to 1120, with an average annual catch of
100
•^S-^rS^V^^:, 456 ± 257. The early and late halves (June vs. July) of
90
80 seasons alsovariedwidelywith respecttosample sizes,
70 -<*>% with mean catches at 259 ± 206 and 197 ± 143, re-
60 spectively. The differences between the earlyand late
50 sample sizes, though large, are not statistically signifi-
40 '"4-8
30 ^ cant bypaired samples £-tests (t = 1.622, df= 39, p =
20 •-gcog-q 0.113), nor by the non-parametric Wilcoxon's signed-
10 ranks test (p = 0.226).
The complete set of catch records, subdivided by
59 62 65 68 71 74 Y77ea8r0 83 86 89 92 95 98 early (A = June) and late (B = July) catches, are sum-
marized by phenotype in Table 1. In the table, the
Fig. 1. ThedeclineinthefrequencyofmelanicB.betulariabe- melanics (f. carbonaria) are separated from the other
ptlwoetetned19f5r9omthJruonueghsa1m9p9l8esat(CsaolliddysCyombmomlos)n,anWdesftroKmirJbyu,lyEsnagmlpalneds, phenotypes (pale = f. typica or"typicals" andinterme-
(opensymbols). diates = f. insularia). The intermediates have re-
mained rare at Caldy Common (for complete data
through 1993 see Clarke et al. 1994), therefore the
combinedcategory(T+I) isessentially"typical" (pale),
hypothesis is thatthere shouldbe no difference in the ornon-melanic.
percentage ofmelanics caught in the early and in the Sample sizes permitting, the numbers of melanics
late samples taken duringthe same summer. and non-melanics collected during the first half and
Laboratory experiments on differential emer- secondhalfofeach seasonwere comparedusing2x2
gence. To determine ifdifferential emergence (time contingencyG-tests ofindependence. The G statistics
to eclosion) ofmelanicversus pale phenotypes exists, arelistedinTable 1. Ofthe38comparisons made, only
we crossed known heterozygous melanic B. betularia three showedsignificantdifferences inthephenotypic
cognataria totheirpale siblings. (Theexpectedratioof proportions between the early and late halves ofthe
pales to melanics among the progeny from such mat- same season; in two instances the melanics increased
ings is 1:1.) The stock material was produced by a significantly(p < 0.05) inthesecondhalfoftheseason,
melanic female crossed to a pale male, both ofwhich andin one instance the melanics declinedverysignifi-
were caught at the same location in Pennsylvania in cantly (p < 0.01) duringthe second halfofthe season.
1996, and the crosses used in our experiments were In35 ofthe 38comparisons, nosignificantdifferences
madeusingtheirprogenywhichemergedinthespring in the proportions ofmelanics between the early and
of1997. late subsampleswithin seasonswere observed.
In all, three sets ofsiblings were crossed, and their To test for the possibility that small but consistent
broods were subdivided to avoid overcrowding. The differences might existwithin seasons (differences too
caterpillars were housed inside plastic "garbage cans" slight to be detected by G-tests), we analyzed the en-
andwere providedacontinuous supplyoffreshleaves tire 40-year record by Wilcoxon's signed-ranks test.
from a single, large Chiswell crab apple tree (Malus The null hypothesis is that the differences in the per-
spp.). The caterpillars pupated "at will" in moistened centages ofmelanics collected during first and second
beddings in the bottoms oftheir containers. The pu- halves ofseasons are random. The data showthat the
pae were then stored in containers lined with moist- percentage of melanics increased during the second
ened paper towels. All storage containers were half of summers 23 times, and decreased 17 times
checked daily to remove newly emerged adults until (Fig. 1), but the differences are not significant by
the experiment was terminated several weeks after Wilcoxons signed-ranks test (p = 0.55).
emergences ceased entirely and no living pupae re- Between 1977 and 1997 the annual incidence of
mained. For each brood, each moth to emerge was melanism declinedrapidlyatCaldyCommon (Fig. 1),
identified by phenotype and sex and the date of its andforseveralyearsduringthis sameperiod, thereap-
emergence. peared to be a short run ofseasons in which the per-
centage of melanics decreased in the second half of
Results
summers comparedtothe firsthalf. However, thepat-
Field studies. From 1959 through 1998, 18,255 tern is not consistent throughout the period ofrapid
Biston betularia were collected near Caldy Common. annual decline in melanism, and the differences in
102 Journalofthe Lepidopterists' Society
Table 2. Earlyandlate emergences from reared broods ofB. intraseasonal differences between the phenotypic
bmeetlualnairciaacnodgpnaaltear(itaypifcraol)mpchreonsostesypeexsp.eTctheedpthoepnortoydpuicced1i:s1trriabtuitoisonosf proportions is consistent with type-I error rates ex-
betweentheearlyandlategroupswithineachbroodarecompared pectedwhen large numbers ofstatistical comparisons
byG-testsofindependence. are made (Sokal & Rohlf 1981). Furthermore, only
two ofthe three observed significant deviations from
Brood Melanics Typicals G
random expectations were skewed in the same direc-
A Early 53 46 tion which further supports that these exceptional
Late 49 36 0.31
years can be attributed to chance. Ofthese three ex-
B Early 30 23 ceptional years (1961, 1978, 1986), only 1986 shows a
Late 16 29 4.37* verypronounced deviation in phenotypic proportions
C Early 27 27 between the early and late halves ofa season, and in
Late 23 30 0.47 this instance the significant decline in the proportion
of melanics occurred during the brief run of intra-
**p < 0.05
seasonal declines in melanism that occurred during
melanic frequencies between first and second halves thoseyears ofthe steepest declines in the annual inci-
of seasons are not significant by Wilcoxons signed- dence ofmelanism. Here, atleast, itseems reasonable
ranks test (p = 0.82). to suggest that selection atthe adult stage ratherthan
Differential emergence. We recorded the emer- developmental differences in emergence schedules
gence date, sexandphenotypes ofall adults produced might account for the drop in melanism within that
by the three crosses ofheterozygous melanics mated season.
to their pale, homozygous siblings. Eclosion patterns Differences in emergence schedules between
withinbroods generallybeginwithone orafewadults melanic and pale peppered moths are slight, at best.
emerging from puparia on the initial day, then the Of the three broods we examined, only one showed
number of daily emergences increases quickly and significantly accelerated emergence of the melanics
peaks a few days later, after which the daily emer- versus their typical siblings. Different broods, no
gences tailoffuntilfinallymanydays mayseparatethe doubt, emerge throughout the normal season ofsev-
stragglers from the main group and from each other. eral months, and ifdevelopmental differences, how-
The null hypothesis ofour experiment is that the dif- everslight, between melanics andpale forms are con-
ference in emergence sequence within broods is not sistent, then, in the absence ofoffsetting selection at
relatedtothecolorphenotypes; therefore, thepropor- the adult stage, we should expect to observe a consis-
tions of melanics and typicals should be the same in tent bias favoring melanics earlier in the summer.
the firsthalfofabroodtoemergeasinthesecondhalf. Clearlythis didnothappenoverthe40yearsofobser-
We divided each brood into early and late groups vations at CaldyCommon.
usingthe median.Allofthe mothsemerginguptoand Our developmental studies in the laboratory em-
includingthe daythe firsthalfofthe total emergences ployed the North American subspecies, B. betularia
for the broodwas reachedwere assigned to the early cognataria, and our field studies centered on the
group, and all ofthe moths emerging after that day Britishsubspecies ofpepperedmoths, B. betulariabe-
were assignedto the late group. Table 2 lists the early tularia. Direct comparisons between the two mustbe
and late emergences byphenotype (melanic and typi- qualified. American "pale" ortypical forms are gener-
cal) for each of the three broods. The numbers of ally much darker than British typicals; however, the
melanics and typicals emerging during the early and melanics (called f. carbonaria in Britain and f. swet-
lateperiods are comparedwithin broods by2 x2 con- taria inAmerica) arephenotypicallyindistinguishable,
tingencyG-tests ofindependence. The G statistics are and are caused by alleles at the same locus (Grant &
alsofistedinthetable. InbroodB, melanics are signif- Clarke in prep.). The melanics, both in America and
icantly more common in the early than in the late Britain, have also shown parallel increases (Owen
group (p < 0.05), butno significant emergence differ- 1962) and decreases (Grant et al. 1995, 1996) in fre-
ences are apparentinthe othertwobroods. quencies associatedwith environmental modifications
related to industrial development and urbanization.
Discussion The reductions in melanism on both continents are
Thedatafrom CaldyCommonprovidelittlesupport nowwidespread (Grantetal. 1998).
for the idea that the different phenotypes of Biston Thephylogeneticrelationship (Rindge 1975, Clarke
betularia fly at different times of the season. That etal. 1993), andthegeneticsofmelanisminthesesub-
three ofthe 38 contingencytests indicated significant species (West 1977, Grant & Clarke inprep.), andthe
Volume 53, Number 3 103
ecological events coincidental to the parallel evolu- Acknowledgments
tionarychangesinphenotype frequencies (Grantetal. WethankJeanButler,WinifredCross, SallyThompson,andAn-
1996, 1998) combine to suggestthatwe are observing gela Urionforassistancewithpopulation samplingatCaldyCom-
the same basicphenomenon inpeppered moth popu- mon, and the Nuffield Foundation for fundingthe fieldwork. We
also thank Annie Harvilicz for assistance in feeding caterpillars.
lations onbothsides oftheAtlantic. Whilewemustre- DeaneBowers,LaurenceCook,TheodoreSargent,WardWatt,and
main cautious in extrapolatingwhatwe learnfrom the Lawrence Wiseman helped us improve this report by their com-
studyofone subspecies to drawconclusions aboutthe mentsonanearlierdraft.Wededicatethispapertothememoryof
LadyFeoClarke.
other, we also recognize that whatwe learn from one
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elimination. We hopewe are makingprogress. 16December1999.
