Retelling Experiments: H.B.D. Kettlewell's Studies of Industrial Melanism in Peppered Moths

H. B. D. Kettlewell's field experiments on industrial melanism in the peppered moth, Biston betularia, have become the best known demonstration of natural selection in action. I argue that textbook accounts routinely portray this research as an example of controlled experimentation, even though this is historically misleading. I examine how idealized accounts of Kettlewell's research have been used by professional biologists and biology teachers. I also respond to some criticisms of David Rudge to my earlier discussions of this case study, and I question Rudge's claims about the importance of purely observational studies for the eventual acceptance and popularization of Kettlewell's explanation for the evolution of industrial melanism.     

Industrial melanism in the moths, Diurnea fagella (Oecophoridae) and Allophyes oxyacanthae (Caradrinidae)

Comparative studies of melanism in the two cryptic moth species, Diurnea fagella (Denis & Schiffermüller) and Allophyes oxyacanthae (L.), have been carried out in southern England and south Wales. Estimates of the relative crypsis of the melanic and typical forms of both these species have been made at a number of sites and these were compared with the melanic frequencies in samples from these sites. These comparisons showed that selective prédation could be a major factor in the variation of melanic frequencies of both of these species. A consideration of the spread of melanism in these species suggests that non-visual selection may favour the melanics of D. fagella in urban areas and that non-visual selection, not closely associated with urban conditions, may be responsible for the restriction of melanics in A. oxyacanthae to Britain. The results for these two species are discussed in relation to investigations of melanism in other moth species.     

Recent history of melanism in American peppered moths

Industrial melanism in peppered moths has been studied most intensively in Britain. The first melanic phenotype (effectively solid black) was recorded near Manchester in 1848. By 1895 about 98% of the specimens near Manchester were melanic, and this once rare phenotype had spread across regions of the country blackened by industrial soot. In rural, unpolluted regions, well away from industrial centers, the pale phenotype (peppered with white and black scales) remained the predominant form. During the latter half of the 20th century, following legislation designed to improve air quality, melanics began to decline in frequency and are now rare where once they had been common. Similar evolutionary changes have occurred elsewhere, but records from outside Britain are fragmentary. We have extended previous surveys of American peppered moth populations and present a composite picture of the recent decline in melanism in northern industrial states-Michigan and Pennsylvania-where melanic phenotypes decreased from more than 90% in 1959 to 6% by 2001. We contrast these changes to the near absence of melanism in a southern state-Virginia-during that same period. As in Britain, the decline in melanism in American peppered moths followed clean air legislation.     

Different genes underlie adaptive melanism in different populations of rock pocket mice

Identifying the genes responsible for adaptation has been an elusive goal in evolutionary biology. Rock pocket mice (Chaetodipus intermedius) provide a useful system for studying the genetics of adaptation: most C. intermedius are light-coloured and live on light-coloured rocks, but in several different geographical regions, C. intermedius are melanic and live on dark-coloured basalt lava, presumably as an adaptation for crypsis. Previous work demonstrated that mutations at the melanocortin-1 receptor gene (Mc1r) are responsible for the dark/light difference in mice from one population in Arizona. Here, we investigate whether melanism has evolved independently in populations of dark C. intermedius from New Mexico, and whether the same or different genes underlie the dark phenotype in mice from these populations compared with the dark mice from Arizona. Seventy-six mice were collected from pairs of dark and light localities representing four different lava flows and adjacent light-coloured rocks; lava flows were separated by 70-750 km. Spectrophotometric analysis of mouse pelage and of rock samples revealed a strong positive association between coat colour and substrate colour. No significant differences were observed in the colour of rocks among the four lava flows, suggesting that mice in these separate populations have experienced similar selection for crypsis. Despite this similarity in environment, melanic mice from the three New Mexico populations were slightly, but significantly, darker than melanic mice from Arizona. The entire Mc1r gene was sequenced in all mice. The previously identified mutations responsible for the light/dark difference in mice from Arizona were absent in all melanic mice from three different populations in New Mexico. Five new Mc1r polymorphisms were observed among mice from New Mexico, but none showed any association with coat colour. These results indicate that adaptive melanism has arisen at least twice in C. intermedius and that these similar phenotypic changes have a different genetic basis.     

Tut-tut Tutt, not so fast. Did Kettlewell really test Tutt's explanation of industrial melanism?

Over the past fifty years a legend has developed surrounding H.B.D. Kettlewell's pioneering research on the phenomenon of industrial melanism as illustrated in the peppered moth, Biston betularia. This legend is particularly pernicious in that it unfairly diminishes Kettlewell's intellectual involvement in the work for which he is most famous (Rudge 2006). One of the most persistent allegations made is that Kettlewell's published work neglects or ignores his debt to J.W. Tutt, identified as the first to propose the explanation of industrial melanism at issue in Kettlewell's original investigations (Owen 1997). The following essay first examines whether J.W. Tutt was indeed the first to explain the phenomenon of industrial melanism in terms of differential bird predation and the long term effects of air pollution, before considering further whether J.W. Tutt's explanation was, in point of fact, the explanation at issue in Kettlewell's investigations.     

Industrial melanism: Do we have the answers?

Entomologists from the late 19th century onwards recognized the evolutionary interest of the association of black forms of the peppered moth with industrialization. They developed a qualitative explanation of the phenomenon involving a change in relative crypsis of the phenotypes due to the blackening of the moth's resting background by air pollution. More recently, ecological geneticists have obtained some estimates of predation by birds and of population parameters such as migration rate. Models incorporating these estimates have explored the ways in which natural selection influences spatial variation and the maintenance of polymorphism. Studies on the peppered moth and some of the many other insects exhibiting industrial melanism have concentrated on the variability and dynamics of adult populations. Recent work which has begun to examine the ecology and behaviour of individuals, complete life cycles, and gene-phenotype relationships, is refining our understanding of this adaptation and also of present-day declines in melanic frequencies in response to falling air pollution.     

Industrial and non-industrial melanism in the peppered moth, Bistort betularia (L.)

Abstract. 1. Estimates of the relative crypsis of the melanic and typical morphs of B.betularia have been made at fifty-two sites in southern England and south Wales and these estimates were compared with melanic frequencies in samples from these sites.
2. The decrease from east to west in the frequency of the melanic form, carbonaria , and increase in the frequency of a second melanic form, insularia , across the southern part of England and Wales is largely independent of changes in smoke concentration, sulphur dioxide concentration or relative crypsis.
3. When all the available information on the distribution of the melanics of B.betularia is considered, sulphur dioxide concentration is correlated with the geographic variation in carbonaria frequency. This, together with evidence of the close relationship between carbonaria crypsis and sulphur dioxide levels, indicates that outside southern England and south Wales, either selective predation, or some direct selective effect of pollutants, is of major importance in determining the variation in carbonaria frequency.
4. Some features of the pattern of the spread of carbonaria in England and Wales during the last century give reasons for expecting non-industrial selective factors to be of greater importance in determining the distribution of melanics of B.betularia in southern England and south Wales than in the rest of Britain.     

Background selection by the peppered moth (Biston betularia Linn.): individual differences

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 betularia (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.     

The Structure of Causal Explanations in Population Biology

The scope of this paper can be clarified by means of a well-known phenomenon that is usually called ‘industrial melanism’: the fact that the melanic form of the peppered moth became dominant in industrial areas in England in the second half of the nineteenth century. Such changes in relative phenotype frequencies are important explananda for population biologists. Apart from trying to explain such changes over time, population biologists also often try to explain differences between populations, e.g. why yellow shell colour is dominant in certain colonies of land snails and almost absent in other colonies. The causal explanations that are given to address such explananda are the objects of analysis in this paper. Our primary aim is to explicate their structure: we want to capture the typical ingredients of causal explanations in population biology, and their organisation. Based on this explication, we discuss how natural selection fits into recent mechanical philosophy of science, and engage in the debate on the nature of evolutionary theory.

     

The understanding of industrial melanism in the peppered moth (Biston betularia) (Lepidoptera: Geometridae)

Melanic polymorphism in B. betularia has been extensively studied. Correlations between high melanic frequency and high levels of air pollution have been demonstrated. Kettlewell and others have shown that differential bird predation has an important effect on the maintenance of the polymorphism, and coefficients of visual selection have been obtained on the assumption that the moth habitually rests on tree trunks. Computer models based on these selective coefficients show that they are not sufficient accurately to explain observed melanic frequencies. Other non-visual selective factors and weak frequency-dependent selection have been invoked to improve fits. Analysis of the resting positions of moths recorded in the wild demonstrates that B. betularia does not usually rest in exposed positions on tree trunks, but rather rests on the underside of branches, on trunks in shaded positions just below major branch joints or on foliate twigs. The results of a pilot selection experiment, while agreeing qualitatively with Kettlewell's results, suggest that fitness estimates that assume trunk-resting are quantitively incorrect. The error is greatest for melanic moths in rural areas. It is suggested that visual selective coefficients based on a true assessment of the resting behaviour of the moths may considerably improve the fit between computer predictions and observed phenotype frequency distributions.     

Changing views on melanic moths

The rapid rise in frequency of melanic morphs in several moth species, especially the peppered moth Biston betukria , in industrial regions during the 19th century, and the subsequent rapid decline, indicate the action of strong selection. There has recently been a tendency to criticise and question all aspects of research on industrial melanism, including the experiments which suggest that selective predation plays an important part in the changes. These experiments are reexamined, together with evidence for changes in appearance of tree surfaces and for relation of initial melanic frequency to subsequent rate of decline. It is suggested that intense pollution may have been required to drive the carbonaria morph to a high frequency, with frequency patterns over a mosaic environment smoothed by migration. Improvements in these extreme locations then triggered the decline, with litde indication of the environmental changes in areas of moderate pollution. Reasons for criticism of past work are discussed. Industrial melanism continues to provide an exceptional opportunity to analyse a pattern of selection and change in gene frequency.     

Immune defence,a possible nonvisual selective factor behind the industrial melanism of moths (Lepidoptera)

It is generally believed that industrial melanism in Lepidoptera is mainly caused by differential predation by birds. In polluted areas, melanic individuals are favoured by natural selection because they are better camouflaged than pale moths on lichen‐free and sooty tree trunks. In this article, we show that, in the black arches moth (Lymantria monacha), melanic morphs have a stronger encapsulation response than pale morphs against nylon monofilament implants. This indicates that the melanic and pale morphs differ in the strength of their immune defence. The same chemical precursors and their end product, melanin pigment, are involved in the encapsulation response and in the external coloration. Thus, it seems that there may be two possible, not mutually exclusive, explanations for the frequency changes observed in the industrial melanism of moths. The dominant gene causes an increase in the amount of melanin pigment and its precursors. This increase causes two changes: an intensified immune defence as a form of improved encapsulation ability of foreign objects, and the well‐known protective dark coloration (a case of relational pleiotropy). It seems possible that industrial melanism is a by‐product of selection on the strength of immunity. In the field, these pleiotropic aspects are exceedingly difficult to distinguish from each other, and the factors may even be compensatory. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 831–838.     

Allelic melanism in American and British peppered moths

Parallel evolutionary changes in the incidence of melanism are well documented in widely geographically separated subspecies of the peppered moth (Biston betularia). The British melanic phenotype (f. carbonaria) and the American melanic phenotype (f. swettaria) are indistinguishable in appearance, and previous genetic analysis has established that both are inherited as autosomal dominants. This report demonstrates through hybridizations of the subspecies and Mendelian testcrosses of melanic progeny that carbonaria and swettaria are phenotypes produced by alleles (isoalleles) at a single locus. The possibility of close linkage at two loci remains, but the simpler one-locus model cannot be rejected in the absence of contrary evidence.     

The peppered moth and industrial melanism: evolution of a natural selection case study

From the outset multiple causes have been suggested for changes in melanic gene frequency in the peppered moth Biston betularia and other industrial melanic moths. These have included higher intrinsic fitness of melanic forms and selective predation for camouflage. The possible existence and origin of heterozygote advantage has been debated. From the 1950s, as a result of experimental evidence, selective predation became the favoured explanation and is undoubtedly the major factor driving the frequency change. However, modelling and monitoring of declining melanic frequencies since the 1970s indicate either that migration rates are much higher than existing direct estimates suggested or else, or in addition, non-visual selection has a role. Recent molecular work on genetics has revealed that the melanic (carbonaria) allele had a single origin in Britain, and that the locus is orthologous to a major wing patterning locus in Heliconius butterflies. New methods of analysis should supply further information on the melanic system and on migration that will complete our understanding of this important example of rapid evolution.     

Theoretical models of melanism in Biston betularia—a review

The various theoretical models that have been constructed to explain the spatial and temporal changes in the frequency of the melanic morph of the moth Biston betularia are reviewed. The assumptions that are made in these models are discussed. It is shown that these models do explain the gross features of the spatial and temporal distribution of melanic variety in England and Wales. The models have been fairly successful in quantitative prediction of the observed decline in the frequencies of the melanic forms by relating selective differentials to the sulphur dioxide levels. Thus these models do yield a reasonable and consistent picture of the gross pattern of changes in melanic frequencies within the limitation of the available data. The models are robust and can thus accommodate changes to the data on the biology and behaviour of the moth. It is shown that neither heterozygote advantage nor non-Darwinian mechanism need to be invoked to understand the observed evolution of melanism in B. betularia.     

A reversible color polyphenism in American peppered moth (Biston betularia cognataria) caterpillars

Insect body color polyphenisms enhance survival by producing crypsis in diverse backgrounds. While color polyphenisms are often indirectly induced by temperature, rearing density, or diet, insects can benefit from immediate crypsis if they evolve polyphenisms directly induced by exposure to the background color, hence immediately deriving protection from predation. Here, we examine such a directly induced color polyphenism in caterpillars of the geometrid peppered moth (Biston betularia). This larval color polyphenism is unrelated to the genetic polymorphism for melanic phenotypes in adult moths. B. betularia caterpillars are generalist feeders and develop body colors that closely match the brown or green twigs of their host plant. We expand on previous studies examining the proximal cues that stimulate color development. Under controlled rearing conditions, we manipulated diets and background reflectance, using both natural and artificial twigs, and show that visual experience has a much stronger effect than does diet in promoting precise color matching. Their induced body color was not a simple response to reflectance or light intensity but instead specifically matched the wavelength of light to which they were exposed. We also show that the potential to change color is retained until the final (sixth) larval instar. Given their broad host range, this directly induced color polyphenism likely provides the caterpillars with strong protection from bird predation.     

The costs of colour: plasticity of melanin pigmentation in an outbreaking polymorphic forest moth

Outside the context of industrial melanism, little is known about the physiological and ecological importance of genetic melanic polymorphisms in moths. Melanin pigments are synthesized from amino acid precursors and should therefore be costly to produce in nitrogen‐limited insects. A genetic melanic polymorphism is present in adult Malacosoma disstria Hübner (Lepidoptera: Lasiocampidae), a widespread forest moth with outbreaking population dynamics. We test the hypotheses that melanin‐based colouration is physiologically costly in M. disstria, that expression of melanin‐based colouration is a plastic trait which varies with population density and nutrition, and that the genetically based melanic phenotype is disadvantaged under nutritionally poor conditions. Two experiments were used to test these hypotheses. A field study compared pigmentation and phenotypic frequencies in moths collected from high‐ and low‐density populations. A laboratory experiment investigated the effects of larval nitrogen availability on adult pigmentation and phenotypic frequencies. High population density and nitrogen limitation reduced pigmentation and size of all moths, but phenotypic frequencies were not affected in either experiment. The effects of diet on both pigmentation and size were stronger for melanic moths than for typical moths. Our results show that adult melanism in M. disstria is physiologically costly, that colour expression is plastic despite its genetic component, and that the melanic phenotype may be disadvantaged under poor conditions but favoured under good conditions. We suggest that temporal variation in selection and trait plasticity help maintain polymorphism stability.     

A Study of the Biological Preferences of Secondary School Pupils in Israel

An interactive computer simulation was designed as an aid to those teaching evolutionary theory. The simulation was inspired by (tie example of industrial melanism in the peppered moth. A BBC microcomputer is used to present a variegated display overlaid with moth shapes, where the colour of some moths is more like that of the background display than others. The student adopts the role of predator, and uses a mouse-pointer to ‘kill’ as many moths as possible in 18 sec. Surviving moths 'breed' to produce the next generation. Since practical trials demonstrated that moths are killed as an inverse function of degree of crypsis, progressive evolution occurs through the generations. The program also stores moth colour frequency data and, at a point chosen by the user, plots histograms for each generation. Development problems, and the simulation's value as a teaching aid, are discussed.     

A bird’s eye view of the peppered moth

Industrial melanism in Biston betularia is one of the best known examples of the role of natural selection in evolution and has received considerable scrutiny for many years. The rise in frequency of the dark form of the moth (carbonaria) and a decrease in the pale form (typica) was the result of differential predation by birds, the melanic form being more cyptic than typica in industrial areas where the tree bark was darkened by air pollution. One important aspect of early work evaluating the relative crypsis of the forms of B. betularia on tree trunks with different lichen flora was the reliance on human observers. Humans, however, do not have the same visual capabilities as birds. Birds have well‐developed ultraviolet (UV) vision, an important component of their colour processing system that affects many aspects of behaviour, including prey detection. We examined the UV characteristics of the two forms of B. betularia and a number of foliose and crustose lichens. In human visible light the speckled form typica appeared cyptic when seen against a background of foliose lichen, whereas the dark form carbonaria was conspicuous. Under UV light the situation was reversed. The foliose lichens absorbed UV and appeared dark as did carbonaria. Typica, however, reflected UV and was conspicuous. Against crustose lichens, typica was less visible than carbonaria in both visible and UV light. These findings are considered in relation to the distribution and recolonization of trees by lichens and the resting behaviour of B. betularia.     

Insect melanism: the molecules matter

Insect melanism, especially in the peppered moth Biston betularia, has long been a textbook case of evolution in action. Hypotheses of the role of natural selection in maintaining melanic polymorphisms have implicated a wide range of explanations in various species, but to understand fully the ecology of melanism, we need to understand its molecular and developmental genetic basis. Because developmental genes often affect more than one trait, identifying the genes responsible for melanism is crucial for a thorough understanding of the fitnesses and selective responses of melanic alleles in nature. Molecular genetic information is also vital for elucidating the evolutionary history and possible mechanistic diversity of melanism among species. Recent studies of the developmental genetics of melanin pigmentation in Drosophila, and of the genetics of pigmentation differences among other insect species, have provided valuable insights into the underpinnings of this important source of polymorphism throughout the Insecta.