Biological Journal o f t h e Linnean Sociely (1988), 33: 217-232. With 5 figures
Background selection by the peppered moth
(Biston betularia Linn.): individual differences
BRUCE GRANT
Department of Biology, The College of William and Mary, Williamsburg, Virginia,
23185, U.S.A.
AND
RO RY J. H O W L E T T
Department of Genetics, University of Cambridge, Downing Street, Cambridge C B 2 3EH
Received 24 April 1987, accepted for publication 3 August 1987
The hypothesis that dimorphically coloured, cryptic moths select appropriate rest sites by
comparing their body scales to substrate reflectance was tested using typical and melanic morphs of
the peppered moth, Biston betuiaria (L.). Experiments designed to block the individual’s inspection
of its inherited colour phenotype do not support Kettlewell’s contrast/conflict (self-inspection)
hypothesis. Instead, tracking of marked moths over successive days revealed individual differences
in rest-site selection which were not related to treatments, experience (imprinting), nor closely to a
moth’s inherited colour pattern. Differences between family broods indicate that some genetic bias
in background selection exists. The production of artificially selected lines with consistent but
opposing preferences will allow us to investigate the co-evolution of pattern and behaviour.
KEY WORDS:-Background
selection
Biston betularia - coadaptation
evolution - industrial melanism - polymorphism supergene.
-
~
contrast/conflict
-
~
CONTENTS
Introduction . . . .
Materials and methods.
.
The scoring pens . .
Behaviour in captivity.
Contrast/conflict . .
Individual differences .
. . . . .
Results
Contrast/conflict . .
Individual differences .
. . . .
Discussion,
Acknowledgements
. .
References. . . . .
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23 1
INTRODUCTION
No example of witnessed evolution is better known to students of biology than
industrial melanism in the peppered moth, Biston betularia. Yet, despite
0024-4066/88/030217
+ 16 $03.00/0
217
0 1988 The Linnean Society of London
218
B. GRANT AND R. J. HOWLETT
continued investigation many questions still remain as to this moth’s ecology, its
behaviour, and the environmental factors responsible for the well-documented
changes in frequency of its typical and melanic colour morphs (Clarke, Mani &
Miynne, 1985; Cook, Mani & Varley, 1986). While several workers have
questioned the extent to which differential predation by birds accounts for the
shifting morph frequencies (Lees & Creed, 1975; Steward, 1977a; and for
reviews, see Jones, 1982, and Howlett & Majerus, 1987), predation continues to
receive the greatest attention in most popular accounts largely because an
impressive body of data demonstrates that conspicuous moths are more likely to
fall victim to predation than do moths that are harder to find (Clarke &
Sheppard, 1966; Kettlewell, 1955a, 1956, 1973; Whittle, Clarke, Sheppard &
Bishop, 1976). This is not to suggest that other factors are not also of importance
(see reviews cited above).
Kettlewell (1955b), in a pioneering effort, tested whether typical and melanic
morphs would actively select appropriate resting backgrounds. By offering
captive moths contrasting backgrounds, he and, later, others (Boardman, Askew
& Cook, 1974; Kettlewell & Conn, 1977) demonstrated that the typicals showed
a significant bias for light backgrounds whereas the melanics took u p positions
on dark surfaces. Elements besides background reflectance are almost certainly
involved in rest-site selection (see review by Howlett & Majerus, 1987), but if
morph-specific reflectance preference does occur, such a behavioural
polymorphism should greatly enhance crypsis.
Just how a moth might decide which background colour to choose has been
the subject of ongoing debate. T o account for how moths could make correct
choices, Kettlewell ( 1955b, 1973) advanced the ‘contrast/conflict’ hypothesis
whereby a moth compares its circumocular tufts of scales to the locally available
backgrounds, coming to rest where the contrast between the substrate and its
scales is least conflicting. (Colour matching of backgrounds has since been
demonstrated in the polymorphic grasshopper, Circotettix rabula, by Gillis, 1982.)
By the mechanism of self-inspection new variants arising in populations would
be behaviourally preadapted to make correct choices given appropriately
heterogeneous backgrounds, thus facilitating the rapid evolution of melanism in
changing environments.
Sargent (1968, 1969) proposed a n alternative to Kettlewell’s hypothesis. He
suggested that differential rest-site selection in polymorphic species, such as
B. betularia, might have a genetic basis. For example, appropriate background
selection might result from a rather improbable pleiotropy of alleles controlling
the colour polymorphism, or, more likely, due to the action of other loci
controlling behaviour i.e. the evolution of a ‘supergene’.
With notable exceptions, few experimental studies have directly investigated
the mechanisms of background selection. In an attempt to test Kettlewell’s
contrast/conflict hypothesis, Sargent ( 1968) painted the head and body scales of
two monomorphic species, Catocala antinympha (a dark noctuid), and Campaea
perlata (a pale geometrid), but as his treatments did not alter preferences, he
concluded that their behaviours were genetically fixed. Unfortunately, however,
by using monomorphic species he missed the point of Kettlewell’s model. Unlike
a polymorphic species whose members, figuratively, need to ask, ‘which colour
am I?’, the evolutionary history of a monomorphic species is more likely to
include strong selection for uniform responses to background reflectance.
BACKGROUND SELECTION IN B I S 7 O X
219
Steward (1976, 1977b, 1985) investigated rest-site selection in a polymorphic
species, Allophyses oxyacanthae. By marking individual moths from broods of
known parentage, he demonstrated individual differences in background
preference. He compared this study with his results using B. betularia, but
unfortunately he did not follow individual behaviour in Biston.
We report here a test of the contrast/conflict hypothesis in polymorphic Biston
betularia, and further, we assess individual differences within and between the
typical and melanic ( = f. carbonaria) colour morphs.
MATERIALS AND METHODS
The Biston betularia we studied were caught near Cambridge, and near West
Kirby on the Wirral (Merseyside) using both M V light traps and assembling
traps. Broods from these collections were also raised in captivity. I n addition, we
include observations on the North American subspecies, B. betularia cognataria
which were captured at the Mountain Lake Biological Station in Virginia. The
melanic polymorphism at this location has been described by West (1977).
The scoring pens
T o determine the moths’ preferences regarding background reflectance, we
put them into scoring pens which were modified versions of Kettlewell’s original
“barrel” experiment. That is, the pens were divided into equal areas of high and
low reflectance, the rationale being that if the moths are indifferent to the
reflectance of the uniformly textured surfaces, they should take up resting
positions randomly with respect to the surfaces available in the pen.
Several pen designs were tried in these experiments, and the results indicated
that the conclusions one might draw from one design sometimes contradict those
derived using a different pen. This could account for the varied results others
have reported in experiments of this sort (e.g. Mikkola, 1984; Howlett &
Majerus, 1987). For example, we learned that the moths react very differently to
the same cloth surface depending on whether the incoming light is reflected
from it or transmitted through it. In addition, if the contrast between
backgrounds is extreme (black us. white), the moths show a strong bias for
black. Thus, the pens we used have undergone an evolution through trial and
error. Except where noted, the bulk of the data reported here were gathered
from the two pen designs described below.
Design A: Four 1-m2 panels were framed to form a cube. Each panel was
divided into two vertical bands producing eight alternating scoring surfaces
( 1 .O x 0.5 m) of stretched, black and grey Irish linen of uniform weave. The
reflectance of the black background as compared to a barium sulphate standard
white was 2.5%, and the grey fabric was 50.4%, as determined by a Macam
Photometrics SMU 101 spot measuring unit. The top of the pen was covered
with a stretched sheet of clear vinyl, and as the sides and the bottom were
shielded, light entered the pen only from above. The experiments were
conducted in deeply shaded wooded areas under protective rain canopies, or in
open-air sheds. One experiment (identified below) was performed in an airconditioned laboratory with dawns and dusks simulated by rheostatically
controlled artificial lighting.
220
B. GRANT AND R. J. HOWLETT
Design B: A large card cylinder (height 1.2 m, diameter 0.8 m) was set up
with alternating stripes (width 25 cm) of black and a heterogeneous 'peppered'
background which was designed to resemble the wings of the typical British
peppered moth (see Fig. 1 ) . T h e pattern was achieved by comparing the mean
reflectance (34.3o;b) and variance (190.2) of painted card to moth wings using
the spot measuring unit (see Howlett & Majerus, 1987). The uniform black
stripes had a reflectance of 5%. The floor of the pen also had equal areas of
these two backgrounds, and the top of the pen was covered with a sheet of clear
glass draped with black muslin which served to diffuse incoming light. Between
trials the pen was rotated 45" to reduce phototactic bias. The experiments using
pen B were conducted in an outside insectory in Cambridge.
Behaviour in captiviQ
During the daylight hours these nocturnal moths remain motionless unless
greatly disturbed, or old, or in poor condition. At dusk, they become active,
walking and/or flying about the pen. They tend to orientate toward the
incorning light. That is, if the light enters from above, they move upwards, but
this can be reversed by covering the top while letting the light enter from below.
Initially, the moths spend much time attempting to escape confinement, but
ultimately, they settle on the walls, or cling to the roof if the surface is
sufficiently textured.
Once settled, most moths pass the remainder of the night without further
exploration. Although virtually motionless, they do not assume the familiar
Figurr. 1 . A sample of the 'peppcred' background usrd in Pen B. T h e mean and variancr o f points
on thc background wcrr matched to the fbrcwings of typical Biston betularia (see text).
BACKGROUND SELECTION IN BZSTON
22 1
daytime resting or ‘sleeping’ postures, but instead appear fully alert with wings
held high, antennae erect, and legs extended. At dawn, they clamp down, often
without moving away from the spot on which they passed the night. The
clamping behaviour resembles a series of ‘push-ups’ in which the moth ‘kneads’
the substrate, and this is accompanied by a slow wing-pumping routine. T h e
whole exercise usually lasts only about 5-10 s.
O n flat, vertical surfaces, moths often rotate the body from side to side while
clamping down, typically ending up with the head pointed upwards. This
behaviour was interpreted by Kettlewell (1973: 71-72) as a searching activity,
but as the moths seldom move far from their original positions, it does not seem
very likely that they are exploring local surfaces at this stage. Moths passing the
night on wooden dowels (imitation branches), reorientate their body axis from
in-line with the dowel (night) to a right-angle to the dowel (day), the branchresting posture described by Mikkola (1984). As they commence the clamping
exercise, the wings d o not make contact with the dowel, but after they rotate the
body, the wings then do make contact and the moth stops in that position,
remaining there until dusk returns. Thus, it seems that the clamping behaviour
is a mechanism which orientates the moth to the configuration of its substrate
rather than an exploratory assessment of local conditions of reflectance.
Many moths do not re-adjust their locations over successive nights. Once
females settle, they tend to remain in place, assuming the alert posture each
night, repeat clamping down again each dawn, until they become old, or mate,
or die. Although active for up to 2 weeks, males too will often remain settled in
one place if not stimulated to move. This suggests that males do not actively fly
about in search of female pheromone, but instead wait for wind-carried
pheromone to reach their out-stretched antennae, and then move toward its
source. However, young, healthy captive moths seldom remain on the floor of
the pen at night when the incoming light is from above. Therefore, to get moths
to make new choices each night, following recording their locations each
morning, they were removed and returned to the floor of the pen before the
start of the next trial.
With the exception of the one experiment conducted indoors, all data
obtained using pen A were scored a t natural dawn so that a given moth made
only one background selection per 24 h period. With pen B, following scoring in
the morning, the moths were returned to the floor, and the pen was draped in
folds of muslin (following Steward, 1976) in a n effort to simulate evening, then
several hours later, gradually unfolded to simulate dawn. Those few moths
making a second selection were recorded and returned to the floor and re-scored
the next morning. The two sexes were never present together in the cylinder so
as to avoid bias due to attraction.
Occasionally moths overlapped the border between two backgrounds. As a
rule of thumb, moths were regarded to be overlapping if the head was within
half a forewing’s width of a border; these were not recorded as having made
clear choices. Moths remaining on the pen floor were also excluded from the
analysis.
Contrastlconjict
I n an effort to ‘fool’ the moths about their natural colours, Sargent’s (1968)
technique of painting scales was attempted. This approach was soon abandoned
B. GRANT AND R. J . HOWLETT
222
because much of the painted body-wool pulled away from the moths when they
became active at night. In other instances, painted moths remained on the pen
floor, indicating the treatment had debilitating effects. As an alternative to
painting, the moths were fitted with white or black paper collars to block their
vision to the rear. The collars (6.5 mm diameter) were about double head width
(3.2 mm) as measured from the outer edges of the eyes (Fig. 2 ) . The head itself
is relatively devoid of scales except for the sparse distribution fringing the eyes,
and small tufts on the palpi; these few remaining scales were touched up with a
water-soluble acrylic paint. When resting normally, untreated moths tuck their
heads ventrally so the unobstructed view includes the undersides of the
antennae, the background surface, and the mass of woolly scales on the upper
thorax and the proximal joints of the forelegs. Because the collars restrict full
head movement, a treated moth at rest is unable to see its wings or its ventral
surface, except for the distal tips of the forelegs. T h e collars did not impede
walking or flight.
Individual dzferences
T o track the behaviour of individual moths over successive days, moths were
marked either by colour-coded dots of paint on the wings, or by fixing
numbered paper tags ( 1 mm) to a forewing with clear nail polish.
RESULTS
Contrastlconjict
Early attempts to test the mechanism of background matching using collared
moths produced ambiguous results. Initially, North American cognatnria were
tested in a black-and-white striped pen. Their strong preference for the black
K g i w 2. White-rullared Bislon betularia if. carbonarza), and black-collared B. betularia rqynalaria
1
f. !ypca'>.
BACKGROUND SELECTION IN BISTOX
223
background was consistent regardless of collar colour (Table 1). T h e following
year the experiments were repeated on British betularia (Wirral) using both
typicals and carbonaria. From the summed data in Table 1, it would appear that
white collars on carbonaria cancel the bias for black stripes shown by uncollared
and black-collared carbonaria. The typicals, however, did not differ significantly
from random expectations for any treatment, although their pooled totals
favour black. Howlett & Majerus (1987) also report a strong bias for black
shown by both morphs when the alternative surface is white.
The ambiguity was finally resolved after individual moths were tracked over
successive choices. Late in the season, several dozen moths were marked, both
collared and uncollared. Of those making a minimum of five scorable choices
(meaning: not overlapping a border, or on the floor), two out of nine whitecollared typicals made all black choices and two others made all white choices
( P = 0.001). Also, two out of six uncollared typicals chose black five times in
succession. From a sample of only 15 moths, the probability of six choosing
either all black or all white given five trials each is P = 0.0002. So, while as a
group the typicals appeared indifferent to background reflectance, individual
tracking revealed significant individual bias.
The following year, a single brood produced by a typical female mated to a
heterozygous carbonaria was tested in the black and grey pen (design A). Each
moth was marked, its position recorded each dawn, and then returned to the
floor of the pen before the next trial. The results are shown in Fig. 3.
In this experiment, conducted indoors, males and females were together in
TABLE
1. T h e total number of choices made by untreated and collared Biston betularia and
B. betularia cognataria in a black and white striped pen. Observations here d o not include
individual tracking, and all moths did not contribute equally to the totals. The G-statistics test
goodness-of-fit to a 1 : 1 within groups, and the heterogeneity G (Het. G ) tests for significant
differences between groups
G-tests, 1 d.f.
Source
Black
cognataria
Melanics
Typicals
White collars
Black collars
Black us. white
Melanic us. typical
betularia
carbonaria
White collars
Black collars
Black us. white
Typicals
White collars
Black collars
Black us. white
Untreated carbonaria vs.
typicals
White
14
40
49
35
N
18
46
57
42
(]:I)
Het. G
5.88*
28.15***
32.78***
20.38***
0.13
0.78
262
75
101
139
90
51
40 I
165
152
116
97
71
98
81
59
214
178
I30
*P<0.05; **P<O.Ol; ***P<O.OOl
38.34
1.37
16.76***
14.25** *
1.52
1.44
1.11
0.0004
7.24**
224
B. GRANT AND K.J . H O W L E T T
............
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Figure 3 . 'l'he pattern of background selections made by individual moths, all siblings, over
succcssive days in pen A. Open symbols indicate grey choices, solid symbols represent black. h
square svmbol indicates a moth was wearing a black collar from that trial forward; a triangle
denotes a white collar. T h e numbers identify individual moths where P = pale (typical) males,
T = pale (typical) females, M = melanic (carbonaria) males, and C = melanic (carbonaria) females.
M a l r s and frmales were together in the same pen. Scc text for details.
the same pen (unlike all other experiments in which the sexes were segregated
into different pens). Mating pairs are not included in the figure. The locations
of males in the pen were probably not independent of local females. Therefore,
considering only the first five choices made by the eleven females, the
probability of five individuals settling exclusively on either black or grey by
chance is P< 0.0003.
What is striking about their behaviour is that background selections are
independent of both moth colour and collar colour, yet moths were clearly not
indifferent to the backgrounds offered. When these individual selections are
pooled, it would appear that the collars have the effect of reversing selections, i.e.
moths tended to rest on backgrounds opposite in colour to their collars! Very
likely, the apparent effect of collaring moths shown in Table 1 was an artefact
produced by collaring moths with varying individual preferences. T h e mere
summation of scores of all moths misses individual differences among the moths,
thus generating spurious conclusions. For this reason, the remainder of the
experiments reported here record the rest sites selected by marked individuals
tracked over successive days.
Individual dzferences
T o assess individual differences as expressed by biases for background
reflectance, uncollared moths, identified by numbered tags, were tested in a
BACKGROUND SELECTION IN BISTON
225
scoring pen (design A) over successive days. Only the first five unambiguous
choices of each moth are reported here; those moths making fewer than five
choices are omitted from this analysis. Choices beyond the first five are also
excluded. The tests include broods raised in captivity from crosses between the
siblings shown in Fig. 3 (males and females tested in separate pens), and males
trapped on the Wirral in June 1986, and male cognataria trapped in Virginia in
August 1986.
Siblings: The background selections from five broods produced from the
matings between siblings are organized in Fig. 4. The ‘mixed broods’ results
(Fig. 4A) are from eggs collected from the mating cages, but the identities of the
several parents are unknown. T h e code-letters listed for the known parents
correspond to those individuals identified in Fig. 3 . Of particular interest would
have been a comparison of the progeny of C-2 (a melanic female which chose
only grey backgrounds) to the progeny of her melanic sister, (2-5, a
predominantly dark-chooser, because both females mated with the same male
(Fig. 4C). Unfortunately, C-5 produced only one son qualifying with five
scorable choices. Of the 11 qualifying male progeny of (2-2, six made all grey
choices five times in a row, and one made five black choices (P<0.000001).
Included among these were five carbonaria males of which four repeatedly
selected only grey ( P < 0.000005).
One female parent was not tested; the remaining broods were produced by
females which showed biases toward black, but as they mated different males,
the comparisons are less meaningful; nevertheless, none of their progeny showed
the striking bias toward grey as did the sons of the grey-choosing C-2.
Although all of the broods were treated alike, eclosing in identical containers,
the possibility of imprinting was explored using the brood from C-4 x P-8; half
of that brood eclosed in a dark-sided container topped by black netting, and the
other half eclosed in a light-sided container covered with white netting. The
results (Fig. 4D) indicate that early experience does not imprint the moths to
corresponding reflectances when selecting rest sites.
Comparisons of morphs, and of sexes, for individual background selections
over all broods appear in Table 2. The expected numbers of individuals are
derived from the binomial (B G) where the probability of a moth selecting
either black or grey is equal (B = G = 0.5). Because of limited sample sizes
within groups, the six classes of black to grey selections, ranging from five
choices of all black ( 5 : O ) to all grey choices ( 0 : 5 ) , are pooled into three
‘phenotypic’ categories defined as follows: black ‘tendency’ = 5 : 0 4 : 1, black
to grey, respectively; ‘Indiscriminate” = 3 : 2 2 : 3; and grey ‘tendency’
= 0 : 5 + 1 : 4. The expected ratio of moths showing black, indiscriminate
or grey tendencies is 3 : 10 : 3 .
These data indicate that no differences exist between the typicals and
carbonaria in the background selections (G, = 1.63, P>0.25), but that the males
and females do differ significantly in their behaviour (G, = 12.63, P<0.005),
with the males biased towards grey, and the females skewed towards black.
+
+
+
Wirral: As all of the broods raised in captivity were originally derived from a
single pair trapped on the Wirral in 1984, the test results may not reflect the
parent population. Therefore, the experiments were repeated using wild-caught
males in 1986. T h e results are also listed in Table 2. Again, the male bias for the
226
B. GRANT AND R. J. HOWLET'I'
C?,
.
A
Ic3
5 3
B
C
Figurr 4. 'l'he pattern of background selections of individual moths from broods produred from
crosscs hrtween several of the siblings identified in Fig. 3. Black symbols indicate black choices, and
light symbols indicate grey. Males and Females were tested in separate pens of design A. Only the
first five scorable choices are recorded here.
grey background is evident, but here there is a significant difference between the
morphs (G, = 6.80, P<0.05). Both morphs include larger numbers of greychoosing individuals than binomial predictions, but there were disproportionately larger numbers of black choosers among the typicals with the
indiscriminate class most noticeably under represented. This difference between
morphs is missed when total choices are compared (Table 2) even though all
moths made exactly the same number of choices.
BACKGROUND SELECTION IN BISTON
227
TABLE
2. The distributions of indiuidual moths making five scorable choices between black and
grey backgrounds (pen A) grouped under Black (5: 0 + 4 : 1, for the number of black and grey
choices, respectively), Indiscriminate = I (3 :2 2 :3), and Grey (1 :4+ 0 : 5). The expected
numbers for statistical tests are based on binomial predictions. See test for details
+
G-tests, 2 d.f.
Source
Fema1es
carbonaria
Typicals
Pooled
Males
Laboratory carbonaria
Wild carbonaria
Pooled carbonaria
Laboratory us. wild
Laboratory typicals
Wild typicals
Pooled typicals
Laboratory us. wild
carbonaria vs. typicals
Laboratory
Wild
Pooled
Females us. Laboratory males
cognataria
Black
I
8
3
13
12
25
11
3
5
8
5
11
16
Grey
3
1
4
26
16
32
58
22
38
x
24
16
40
45
59
I04
16
12
33
15
31
18
44
30
77
(3 : 10: 31,
Het. G
3.40
10.19**
13.23**
23.23***
5.64
16.23***
19.90** *
1.97
1.63
6.80*
7.94*
12.63* * *
7
15
2
24
*IJ<o.o5; **P<O.Ol; ***P<O.OOl.
In general, the distributions of choices made by the wild-caught males and
the laboratory-raised males are very similar. Statistical comparisons show no
significant differences between the two sources of carbonaria, nor between the two
sources of the typicals, whether the comparison is made on individual
(binomial) scores or on summed data. A comparison between morphs using
group sums fails to indicate any difference between the number of black versus
grey choices made by the carbonaria and typicals, although, as noted above, there
are more predominantly black-choosing individuals among the typicals.
Subspecies cognutaria: The general reaction of a small sample of wild-caught
North American cognutaria to the backgrounds offered in pen A was rather the
reverse of betularia. Although too few moths qualified with the minimum of five
scorable choices for a binomial analysis, there were more predominantly black
choosers than grey choosers among them. The total number of black choices
made by cognataria does not differ significantly from random expectations
(GI = 2.14, P>O.l), but does differ significantly from wild-caught betuturia, i.e.
the distributions between the two subspecies appear heterogeneous ( G I = 8.72,
P < 0.005).
The results in Table 4 record the responses of Cambridge B . betularia in pen B.
Wild-caught moths, and those from a single laboratory brood, were pooled after
determining homogeneity by G tests. Only the first seven unambiguous choices
are summed within groups so that each moth contributes equally. TWO
differences are immediately obvious from the results in Table 3 (design A ) .
B. GRANT AND R. J. HOWLETT
228
TABLE
3. A summary of total choices made by wild (caught on the Wirral) and lab (progeny from
sibling crosses raised in captivity) Biston betularia, and wild B. betularia cognataria tested in pen A.
All moths contributed equally (five choices each) to the totals presented here
~
~
G-tests, 1d.f.
Sourcr
..
~
__
Fcmalcs
cnrhonarin
'Iypicals
Poolrd
klaic:
Laboratory carbonaria
h X d mrbonaria
Pooled caThfffZaria
Lab Z ~ A .wild
Lab typicals
IViId typicals
Pooled typicals
Lab us. wild
carbonaria 1-J. typicals
Laboratory
\Vild
Pooled
Black
Grey
.a-
72
45
117
48
35
83
120
80
200
83
124
207
142
171
313
225
295
520
15.65** *
7.52
2 1.76***
66
91
157
99
129
228
165
220
358
6.64**
6.60*
13.17***
(1 : I )
Het. G
4.83*
1.25
5.81*
1.41
0.07
0.39
0.02
0.09
Fcmalcs oz. males
Laboratory carbonaria
Lab typirals
16.93***
5.74*
68
cognalaria
cs. wild males
52
120
2. I4
8.72**
*P<0.05; **P<O.OI; ***P<0.001
Firstly, there is no significant difference between the sexes within the typical
morph. Unfortunately too few carbonaria were available for study. Secondly,
there was a significant difference between the morphs. However, as in the
previous experiment, there was evidence of individual heterogeneity within
T A B L E 4. A summary of total choices made by Biston betularia (Cambridge) tested in pen B. All
moths contributed equally (seven choices each) to the totals presented here. The G-statistics test
goodness-of-fit to a 1 : 1 within groups, and the heterogeneity G (Het. C) tests for differences
between groups.
G-tests, 1 d.f
-~
Sollrcr
Jv
Black
Peppered
32
47
17
49
58
105
( I : 1)
Het. G
--_
~~
Males
carbonaria
'I'ypicals
carbonaria
2's.
4.67*
1.15
typicals
Frmalc typicals
Frinalcs L'S. typical males
Pooled typicals us. carbonaria
5.72*
44
82
126
11.64***
2.32
Il.OO***
BACKGROUND SELECTION IN BISTOX
229
morphs and the sample size was low. It is therefore too early to conclude that
there is a behavioural polymorphism associated with the colour pattern
polymorphism.
DISCUSSION
Any experiment using captive animals to learn about their behaviour is
subject to the familiar criticism that organisms removed from their natural
setting probably behave abnormally. The presence of other moths and the
restriction of free movement in an enclosure undoubtedly must modify
behaviour. The selection of resting sites by Biston betularia almost certainly
involves cues in addition to background reflectance. An assessment of this
complex behavioural phenotype is difficult without identifying the role of the
various factors and their possible interaction in rest-site selection. Work in
progress is aimed at remedying this deficiency in our knowledge.
Nevertheless, the moths were not indifferent to the backgrounds offered them
in captivity, nor did all individuals respond to the same backgrounds in like
fashion. The most obvious difference between the backgrounds used was light
reflectance. The alternatives offered in pen A were of identical weave so surface
texture would not bias the outcome. Furthermore, the moths responded
differently to the same weaves under different lighting arrangements (reflected
versus transmitted). Because the moths take up their resting positions well before
the sun breaks the horizon, heat reflectance from the thin fabric panels probably
did not differ appreciably from ambient air temperature. But even if other
factors besides light reflectance confounded the experiments, individual moths,
and those from different families, different sexes, and the cognataria race,
responded differently to the available cues. The results using pen B, despite
obvious differences in experimental design and in sample sizes, are generally
consistent with those obtained using pen A.
Our experiments provide evidence against the contrast/conflict (selfinspection) hypothesis advanced by Kettlewell, a t least in B. betularia. The
observation that significant individual differences in background preference
exist within morphs is incompatible with the contrast/conflict model.
Furthermore, differences observed among the broods produced from sibling
matings indicate that a genetic bias in background selection exists. This
conclusion is also supported by the failure to modify behaviour by attempts a t
imprinting, and collaring.
If the preferences for different backgrounds are influenced by heredity, then
non-random associations, either by linkage or expression, producing harmonious
combinations of morph type and behaviour, should promote the evolution of a
‘supergene’. While there is an obvious advantage to moths which rest on correct
backgrounds, moths which actively select inappropriate backgrounds are
inherently prone to fall victim to predators. However, the observation that
different individuals within a morph have consistent but opposing preferences
might mean that a supergene has not had sufficient time to evolve. A similar
hypothesis has previously been invoked to explain the failure to demonstrate
morph-specific background preference in the North American geometrid,
Phigalia titea, (Sargent, 1969). For B. betularia the possibility that the supergene
is in the process of breaking down should not be overlooked. This is especially so
230
B. GRANT AND R. J. HOWLETT
given that the frequency of the carbonaria morph is presently undergoing a rapid
decline in many parts of Britain (Clarke et al., 1985; Cook et al., 1986; Howlett
& Majerus, 1987).
Until the genetic basis of background selection is determined with certainty,
further discussion of possible coadapted supergenes must remain speculative.
Therefore, we are continuing to explore the role of genes in this behaviour
through two-way directional selection in a n attempt to establish dark-choosing
and light-choosing lines. I n addition, cognataria and both morphs of betularia
have been hybridized, and these, as well as their backcrosses and F, generations,
will be measured for reflectance preferences to determine if genetic differences
between the races contribute to their different background selection behaviours.
If a supergene in a state of flux is not involved, then we can think of no
obvious reason for a moth to show a n active preference for an inappropriate
background. It is impossible to answer this question until the natural rest sites of
this species are known with certainty.
It has traditionally been supposed that the typical morph gained protection
from its cryptic resemblance to grey foliose lichens, and that the decline of this
rnorph in polluted areas is related to the concomitant demise of these lichens. If
this causal relationship is true, then the prediction is that lichen should precede
the recovery of the typical morph as the common form. T h a t is, the hiding
Figure 5. Birton betularia typicals and carbonarm posed on the bark of silver birch trecc (Retula
pendufa).Two moths, one of each form, arc on each trunk.
BACKGROUND SELECTION IN BIS’TOX
23 1
places should recover before the hider. But, this is clearly not the case in at least
two regions where the recovery of typicals has been especially well documented
in the virtual absence of these lichens: on the Wirral (Clarke et al., 1985;
personal observations by B.G. in 1984-1986), and in East Anglia (Howlett &
Majerus, 1987; personal observation by R.J.H.).
If changes in the abundance of grey lichens are not prerequisite to shifting
morph frequencies in B . betularia, what other factors might be involved? Clarke
et al. (1985) discuss various possibilities in addition to lichen, and comment on
the gradual lightening of trees in the absence of industrial soot. We would
expand on their observations by noting the striking succession of silver birch
(Betula pendula) on the Wirral diring the last several decades (Ranger Andrew
Brookband, Royden Park, personal communication) and elsewhere in Britain
(see Marrs, Hicks & Fuller, 1986). No close relationship between birch and this
moth has been established to our knowledge, although a historic association has
been suggested previously (see Boardman, et al., 1974). Following Kettlewell’s
lead of posing moths on candidate rest sites, we demonstrate in Fig. 5 that both
morphs might be well concealed on such a mosaic background.
It is certainly possible that no single ecological factor is solely responsible for
the shifting polymorphism in B. betularia (see Jones, 1982). Only through
continued, intensive field studies are we likely to gain insight as to this moth’s
habits in nature. And, only through controlled crosses and appropriately
designed tests of behaviour are we likely to gain insight as to the role of genes in
that behaviour. As Biston betularia has served as a paradigm of evolution, it
demands the closest possible scrutiny.
ACKNOWLEDGEMENTS
Although we worked independently, we are indebted to many people who
have helped either one or both of us in ways for too numerous to list here.
Without their assistance, our work would have been very much more difficult.
We gratefully acknowledge: Dr J. Barrett, Mr Andrew Brookbank, Mrs Riki
Butler, Mr & Mrs G. Cross, Dr M. E. N. Majerus, Professor J. J. Murray, Dr P.
O’Donald, Mrs Jewel Thomas, Mr I . P. M. Tomlinson, Mrs Angela Urion, Dr
D. A. West, and Mr G. Wynne.
B. Grant is especially grateful to Sir Cyril & Lady Clarke for their uncommon
hospitality during the two summers he worked at their home on the Wirral and
as a guest research worker at the Department of Genetics, University of
Liverpool.
B. G. was supported, in part, by a Pratt Research Fellowship awarded
through the Mountain Lake Biological Station of the University of Virginia
(1983), and by a summer research grant (1984) and minor equipment grant
(1986) from the College of William & Mary. R. J. Howlett was funded by an
S.E.R.C. Quota award.
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