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Industrial Melanism in British Peppered Moths Has a Singular and Recent Mutational Origin

Science  20 May 2011:
Vol. 332, Issue 6032, pp. 958-960
DOI: 10.1126/science.1203043

Abstract

The rapid spread of a novel black form (known as carbonaria) of the peppered moth Biston betularia in 19th-century Britain is a textbook example of how an altered environment may produce morphological adaptation through genetic change. However, the underlying genetic basis of the difference between the wild-type (light-colored) and carbonaria forms has remained unknown. We have genetically mapped the carbonaria morph to a 200-kilobase region orthologous to a segment of silkworm chromosome 17 and show that there is only one core sequence variant associated with the carbonaria morph, carrying a signature of recent strong selection. The carbonaria region coincides with major wing-patterning loci in other lepidopteran systems, suggesting the existence of basal color-patterning regulators in this region.

Industrial melanism in the peppered moth (Biston betularia) is one of the most widely recognized examples of contemporary evolutionary change (13), but beyond the fact that it is a single-locus dominant allele (4), the genetic basis and developmental mechanism distinguishing the black (carbonaria) form from the wild-type [typical (typica)] form are unknown. This knowledge gap undermines our understanding of a comprehensive example of adaptation encompassing the mutational source of a morphological novelty, its mode of action through development, the agent of selection, and the documented spread of the trait in a natural population. Because melanism is widespread in Lepidoptera, and industrial melanism has arisen in many species (1, 5), reconstructing the genetic origins of melanic forms is likely to yield insight into the role of developmental constraint and the importance of “hotspot genes” to morphological evolution (6). Moreover, it is uncertain whether peppered moth industrial melanism in Britain was due to a single recent mutation or to different mutations of varying age. Nineteenth-century records suggest that the spread of the carbonaria morph across Britain emanated from a single point source in greater Manchester, but do not establish a single mutational origin (1), particularly in view of anectodal evidence for recurrent mutation to melanic forms in B. betularia.

In vertebrates, melanism is often associated with mutations within the melanocortin-1-receptor gene (7). In insects, although melanin biosynthesis is well understood in terms of enzyme-mediated substrate transformations (8), the mechanism of regulation is far more diverse, most commonly involving modification of cis-regulatory elements of key genes in the melanization pathway, such as ebony, tan, and yellow (9). The extent of this regulatory diversity is underscored by our recent finding that in B. betularia, the carbonaria morph is not associated with molecular variation within any of the canonical melanization pathway genes (10). This suggests that the carbonaria phenotype is controlled by an undescribed developmental switch with major phenotypic effects.

The failure of a candidate gene approach to identify the genetic basis for melanism led to the construction of a linkage map to identify the chromosomal region containing the locus controlling the carbonaria-typical polymorphism, and to examine the molecular evidence for the hypothesis that the carbonaria morph in the United Kingdom is descended from a single mutation of recent origin. The carbonaria morph mapped to a linkage group (LG) that is orthologous to Bombyx mori chromosome 17 in the main mapping family and in a second, independent, carbonaria-typical cross, added to construct a denser combined map for LG17 (Fig. 1) (11). The identity of the “carbonaria chromosome” was confirmed with the use of fluorescence in situ hybridization (FISH) (11) (fig. S1). The linkage and physical maps show complete shared synteny to Bombyx mori chromosome 17 (Fig. 1); however, the physical positions of genes are much more similar to those in the Bombyx mori chromosome than to those in the B. betularia recombinational map. In particular, the positioning of sulfamidase at the very top of the painted chromosome and chitinase just below it revealed that the upper half of LG17 is deformed by the large gap between rpl38 and myosin HC, which has the effect of shifting the carbonaria locus downward. This gap was present in both mapping families, suggesting that the discrepancy between physical and centimorgan distances is due to a recombination hotspot near the upper end of the chromosome. The core region under investigation displays normal recombination dynamics.

Fig. 1

Chromosomal localization of the carbonaria locus and synteny between B. betularia and Bombyx mori chromosome 17. The recombinational positions of genes mapped to B. betularia linkage group 17 (LG17) are related to their physical positions in Bombyx mori chromosome 17 (righthand vertical bar) by interconnecting lines. A scheme of bacterial artificial chromosome (BAC)–FISH–painted B. betularia chromosome 17 (lefthand vertical bar) shows the physical positions of seven loci (derived from the FISH-painted chromosomes 17 presented in fig. S2). The region in Bombyx mori indicated as carb is defined by heat repeat-containing protein and proteasome 26S non-ATPase subunit 4, which are located on the fluorescent carbonaria BAC on the B. betularia chromosomes. A 1.4-Mb BAC tilepath centered on the carbonaria locus (black box within LG17) is presented in more detail in Fig. 2.

To determine whether the same core haplotype distinguishes all UK carbonaria morphs, we conducted population genetic surveys at two spatial scales: one on a sample of B. betularia males caught in 2002 from two sites close to Leeds (32 carbonaria and 32 typicals), and one on a larger UK-wide sample (78 sites) spanning the period 1925–2009 (median year of collection = 1974) (fig. S3 and table S4). Both samples were scored for single-nucleotide polymorphisms (SNPs) at six widely spaced loci within the region (additional closely linked polymorphisms corroborated allele identities at these test loci, collectively defining “mini haplotypes”). In the Leeds sample, the distribution of SNP genotypes among morphs (Fig. 2) revealed significant morph-genotype associations (PMG) at loci a, b, c, and d, with weaker associations at e and f. A similar pattern of association was detected in the UK-wide sample (fig. S4). Most notably, across both samples the A/A genotype at locus c is absent in all 97 carbonaria specimens, effectively establishing that all carbonaria morphs carry a G at this position (the A in G/A heterozygotes is presumed to be carried on the typica haplotype). This 100% association leads to the conclusion not only that the same chromosomal region (locus) underlies the genetic determination of all carbonaria in the sample but that all carbonaria in the United Kingdom are derived from a single ancestral haplotype. Our inference is additionally supported by the rarity of carbonaria morphs with the C/C genotype at locus d (A/A for locus e was also rare in the Leeds sample), implying that the ancestral carbonaria haplotype was defined by the alternative alleles, G and G, at loci c and d, respectively. Clearly, however, given the occurrence of the G allele at locus c in typicals, this cannot be the actual morph locus.

Fig. 2

Association between morph type and SNP genotype at six marker loci within the carbonaria region in a sample of wild B. betularia from Leeds in 2002. A chromosome section, approximately 1.4 Mb in length, is represented as a horizontal line along which the relative positions of the genetic markers (a to f) and 10 genes (boxes) are indicated (gene abbreviations are as follows: WD40 repeat domain 85, cyclin-dependent kinase 2, enoyl-CoA hydratase precursor 1, heat repeat-containing protein, and proteasome 26S non-ATPase subunit 4). For each marker locus, the correspondingly labeled bar chart (a to f) shows the frequency distribution of SNP genotypes, separately for carbonaria (black bars) and typical (stippled bars) samples. PMG, the probability of the observed pattern of association between morph and genotype occurring by chance, and estimates of D′, a standardized measure of linkage disequilibrium, are also presented for each marker locus. Marker positions are approximations based on the the size distribution of BACs in our library and the distances between orthologous genes in Bombyx mori.

The pattern of morph-genotype association for typicals is more complex than for carbonaria (Fig. 2 and fig. S4). In particular, at loci c to f, the high frequency of typicals that are homozygous for the carbonaria-type marker allele (the leftmost genotype class) contrasts with the relative absence of carbonaria that are homozygous for the typical-type marker allele (the rightmost genotype class). At loci a and b, however, typicals homozygous for the carbonaria-type marker allele are completely or nearly absent (these typica haplotypes do occur in heterozygotes at both loci in both samples). The likely explanation for this pattern is that the ancestral carbonaria haplotype arose by mutation from a typica haplotype, so that many polymorphisms in the region will not be unique to carbonaria (that is, high frequencies of typicals either heterozygous or homozygous for the marker allele coupled to carbonaria at loci c to f). By chance, some variants defining the progenitor carbonaria haplotype were uncommon in the ancestral typical population (for example, at loci a and b). Thus, the marker allele frequencies we now see in the typical population reflect both what was originally present and what has been introduced via the spread and introgression of the carbonaria haplotype.

In Fig. 2 (and fig. S4), the dominance of the carbonaria allele obscures the true frequencies of morph-marker haplotypes by masking the presence of typica haplotypes in carbonaria/typica heterozygotes (phenotypically carbonaria). Reconstruction of the core multilocus haplotype frequencies for carbonaria and typica alleles (Table 1) clarifies the identity of the ancestral carbonaria haplotype within a more diverse population of typica haplotypes. The ancestral carbonaria haplotype defines the coupling phase for the carbonaria allele, as well as the repulsion phase composed of the alternative SNP alleles at each marker locus, generated by recombination (which is also taken to be the coupling phase for typica).

Table 1

Frequency of reconstructed core haplotypes carrying carbonaria and typica alleles. Multilocus haplotypes were defined for three (b, c, and d) of the six marker loci. Locus positions are shown in Fig. 2. The inferred six-locus haplotype of the original carbonaria mutant is CAGGTA (on the upper strand of the schematic sequence in Fig. 2).

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We assessed the degree of nonrandom association between the morph and marker alleles with a standardized measure of linkage disequilibrium, D′ (12), ranging between values of 0 (a fully random association) to 1 (a complete association between allele pairs). Because the underlying model assumes random mating within the population from which the sample was taken, it is only appropriate to calculate this statistic for the Leeds sample. D′ was high (>0.5) for all markers across the 1.4-Mb sequence examined, defining a region of approximately 400 kb where D′ > 0.9, with a maximum D′ at locus c (Fig. 2). Interpolation of D′ values at loci b, c, and d suggests that the genetic polymorphism controlling the morph phenotype is located within 100 kb on either side of locus c. Further consideration of the “missing” (repulsion-phase) haplotypes separately for carbonaria and typica (Table 2) emphasizes the deficit of repulsion-phase carbonaria haplotypes across all six loci. In contrast, typica haplotypes show only weak deficits of carbonaria-type marker alleles, which is consistent with the view that these alleles were segregating in the population before the genesis of carbonaria. At loci a and b, the deficit for typica is greater because the carbonaria-type alleles (C and A, respectively) were rare in the ancestral population. These two loci suggest that carbonaria-to-typica haplotype introgression has been weak.

Table 2

Deficiency of repulsion-phase morph-marker haplotypes. The ratio of observed repulsion-phase (recombinant) haplotypes to their expected frequency if morph and marker alleles were randomly associated, for each morph allele (carbonaria and typica) and marker locus in the Leeds sample, is shown. For each morph allele, the identity of the repulsion-phase marker allele is indicated in parentheses. Locus positions are shown in Fig. 2. P values for a Χ2 test with 1 d.f. are indicated by asterisks: *P < 0.5, **P < 0.01, and ***P < 0.001; n.s., not significant.

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Bombyx mori chromosome 17 and its orthologs in other lepidopterans are rich in major color-patterning genes, such as black moth and wild wing spot (13). These Bombyx mori genes do not map closely to the carbonaria locus (fig. S5). However, Bicyclus anynana LG17 contains two pigment-patterning mutants, 067 and Bigeye, that both affect eyespot size, with Bigeye predicted to reside within the carbonaria region (14). The Bigeye and carbonaria phenotypes are clearly very different, but they share a large increase in the proportion of melanized scales. The carbonaria core region also overlaps the mimetic patterning locus in four Heliconius species, collectively referred to as the Yb-P-Yb/Sb-Cr locus (1517). The B. betularia genes identified in this region so far correspond entirely with those described for the Yb-P-Yb/Sb-Cr region. A major feature distinguishing Heliconius forms is the amount and distribution of black, as with the various B. betularia morphs (4). This unlikely coincidence suggests that the control of melanin pattern formation in these deeply diverged lepidopterans may have a common genetic basis, the functional units of which have yet to be identified.

The rapid spread of an initially unique haplotype, driven by strong positive selection, is expected to generate the profile of linkage disequilibrium we have observed (18), establishing that UK industrial melanism in the peppered moth was seeded by a single recent mutation that spread to most parts of mainland Britain and also colonized the Isle of Man (fig. S4). Paradoxically, although the carbonaria morph is now strongly disadvantageous and consequently rare in the United Kingdom, the rapidity of its decline (19) has minimized the eroding effect of typica introgression on the molecular footprint of strongly positive selection created during its ascendency.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1203043/DC1

Materials and Methods

Figs. S1 to S5

Tables S1 to S4

References

References and Notes

  1. Materials and additional results are available as supporting material on Science Online.
  2. Acknowledgments: This work was funded by Natural Environment Research Council grant NE/C003101/1 to I.J.S. Cytogenetic experiments were financed by Grant Agency of the Academy of Sciences of the Czech Republic grant IAA600960925, Entomology Institute project Z50070508, and Grant Agency of the Czech Republic 521/08/H042 grants to F.M. and M.D. Specimens for the UK-wide survey were made available by several museums and private collectors, listed in table S4. J. Delf produced fig. S3. GenBank accession numbers are listed in table S1.
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