The industrial melanism mutation in British peppered moths is a transposable element.

Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of 'jumping genes' as a source of major phenotypic novelty.

Recessive Z-linked lethals and the retention of haplotype diversity in a captive butterfly population.

Sex chromosomes are predicted to harbour elevated levels of sexually antagonistic variation due to asymmetries in the heritability of recessive traits in the homogametic versus heterogametic sex. This evolutionary dynamic may manifest as high recessive load specifically affecting the homogametic sex, and the retention of haplotype diversity in small populations. We tested the hypothesis that the Z chromosome in the butterfly Bicyclus anynana carries a high inbred load for male fertility and viability. Homozygosity of Z chromosome blocks was produced by daughter-father backcrosses, and inferred from marker loci positioned via a linkage map. Male sterility was, in general, unrelated to homozygosity in any region of the Z, but there was an extreme deficit of homozygous males within a 2 cM interval in all families. In contrast, no corresponding skew in Z genotype was detected in their (hemizygous) sisters. The same pattern was observed in historically inbred lines, indicating a high frequency of recessive lethals in the ancestral population. Allele-frequency changes between 1993 and 2006 (70 generations at Ne ~ 160) show that, despite the loss of many haplotypes, diversity was retained significantly above the neutral expectation. Effective overdominance in the lethal region can account for this effect locally but not in other parts of the chromosome, that are also associated with persistent linkage disequilibrium. These unexpected patterns suggest the operation of other factors, such as epistatic selection, recombination suppression, assortative mating and meiotic drive. Our results highlight the role of balancing selection in maintaining the inbred load and linked genetic diversity.

Genetic convergence of industrial melanism in three geometrid moths.

The rise of dark (melanic) forms of many species of moth in heavily coal-polluted areas of nineteenth- and twentieth-century Britain, and their post-1970s fall, point to a common selective pressure (camouflage against bird predators) acting at the community level. The extent to which this convergent phenotypic response relied on similar genetic and developmental mechanisms is unknown. We examine this problem by testing the hypothesis that the locus controlling melanism in Phigalia pilosaria and Odontopera bidentata, two species of geometrid moth that showed strong associations between melanism and coal pollution, is the same as that controlling melanism in Biston betularia, previously identified as the gene cortex. Comparative linkage mapping using family material supports the hypothesis for both species, indicating a deeply conserved developmental mechanism for melanism involving cortex. However, in contrast to the strong selective sweep signature seen in British B. betularia, no significant association was detected between cortex-region markers and melanic morphs in wild-caught samples of P. pilosaria and O. bidentata, implying much older, or diverse, origins of melanic morph alleles in these latter species.

Evolutionary trajectory of phenological escape in a flowering plant: Mechanistic insights from bidirectional avoidance of butterfly egg-laying pressure.

Phenological escape, whereby species alter the timing of life-history events to avoid seasonal antagonists, is usually analyzed either as a potential evolutionary outcome given current selection coefficients, or as a realized outcome in response to known enemies. We here gain mechanistic insights into the evolutionary trajectory of phenological escape in the brassicaceous herb Cardamine pratensis, by comparing the flowering schedules of two sympatric ecotypes in different stages of a disruptive response to egg-laying pressure imposed by the pierid butterfly Anthocharis cardamines, whose larvae are pre-dispersal seed predators (reducing realized fecundity by ~70%). When the focal point of highest intensity selection (peak egg-laying) occurs early in the flowering schedule, selection for late flowering dependent on reduced egg-laying combined with selection for early flowering dependent on reduced predator survival results in a symmetrical bimodal flowering curve; when the focal point occurs late, an asymmetrical flowering curve results with a large early flowering mode due to selection for reduced egg-laying augmented by selection for infested plants to outrun larval development and dehisce prior to seed-pod consumption. Unequal selection pressures on high and low fecundity ramets, due to asynchronous flowering and morphologically targeted (size-dependent) egg-laying, constrain phenological escape, with bimodal flowering evolving primarily in response to disruptive selection on high fecundity phenotypes. These results emphasize the importance of analyzing variation in selection coefficients among morphological phenotypes over the entire flowering schedule to predict how populations will evolve in response to altered phenologies resulting from climate change.

Zygosity-based sex determination in a butterfly drives hypervariability of Masculinizer.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene (BaMasc) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

The genome sequence of the Squinting Bush Brown, Bicyclus anynana (Butler, 1879).

We present a genome assembly from an individual female Bicyclus anynana (the Squinting Bush Brown; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 457.2 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.1 kilobases in length.

The genome sequence of the Rock Grayling, Hipparchia semele (Linnaeus, 1758).

We present a genome assembly from an individual female Hipparchia semele (the Rock Grayling; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 403.4 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.22 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,540 protein coding genes.

The peppered moth Biston betularia.

Hannah Rowland and colleagues introduce the peppered moth whose industrial melanism was an early evidence for evolution.

Recovering individual haplotypes and a contiguous genome assembly from pooled long-read sequencing of the diamondback moth (Lepidoptera: Plutellidae).

The assembly of divergent haplotypes using noisy long-read data presents a challenge to the reconstruction of haploid genome assemblies, due to overlapping distributions of technical sequencing error, intralocus genetic variation, and interlocus similarity within these data. Here, we present a comparative analysis of assembly algorithms representing overlap-layout-consensus, repeat graph, and de Bruijn graph methods. We examine how postprocessing strategies attempting to reduce redundant heterozygosity interact with the choice of initial assembly algorithm and ultimately produce a series of chromosome-level assemblies for an agricultural pest, the diamondback moth, Plutella xylostella (L.). We compare evaluation methods and show that BUSCO analyses may overestimate haplotig removal processing in long-read draft genomes, in comparison to a k-mer method. We discuss the trade-offs inherent in assembly algorithm and curation choices and suggest that "best practice" is research question dependent. We demonstrate a link between allelic divergence and allele-derived contig redundancy in final genome assemblies and document the patterns of coding and noncoding diversity between redundant sequences. We also document a link between an excess of nonsynonymous polymorphism and haplotigs that are unresolved by assembly or postassembly algorithms. Finally, we discuss how this phenomenon may have relevance for the usage of noisy long-read genome assemblies in comparative genomics.

Selection and gene flow on a diminishing cline of melanic peppered moths.

Historical datasets documenting changes to gene frequency clines are extremely rare but provide a powerful means of assessing the strength and relative roles of natural selection and gene flow. In 19th century Britain, blackening of the environment by the coal-fired manufacturing industry gave rise to a steep cline in the frequency of the black (carbonaria) morph of the peppered moth (Biston betularia) across northwest England and north Wales. The carbonaria morph has declined across the region following 1960s legislation to improve air quality, but the cline had not been comprehensively described since the early 1970s. We have quantified changes to the cline as of 2002, equivalent to an interval of 30 generations, and find that a cline still exists but that it is much shallower and shifted eastward. Joint estimation of the dominant fitness cost of carbonaria and dispersal parameters consistent with the observed cline change indicate that selection against carbonaria is very strong across the landscape (s approximately 0.2), and that dispersal is much greater than previously assumed. The high dispersal estimate is further supported by the weak pattern of genetic isolation by distance at microsatellite loci, and it implies that in addition to adult dispersal, wind-dispersed first instar larvae also contribute to lifetime dispersal. The historical perspective afforded by this study of cline reversal provides new insight into the factors contributing to gene frequency change in this species, and it serves to illustrate that, even under conditions of high dispersal and strong reverse selection acting against it, complete erosion of an established cline requires many generations.

Wing morphological responses to latitude and colonisation in a range expanding butterfly.

Populations undergoing rapid climate-driven range expansion experience distinct selection regimes dominated both by increased dispersal at the leading edges and steep environmental gradients. Characterisation of traits associated with such expansions provides insight into the selection pressures and evolutionary constraints that shape demographic and evolutionary responses. Here we investigate patterns in three components of wing morphology (size, shape, colour) often linked to dispersal ability and thermoregulation, along latitudinal gradients of range expansion in the Speckled Wood butterfly (Pararge aegeria) in Britain (two regions of expansion in England and Scotland). We measured 774 males from 54 sites spanning 799 km with a 10-year mean average temperature gradient of 4 °C. A geometric morphometric method was used to investigate variation in size and shape of forewings and hindwings; colour, pattern, and contrast of the wings were examined using a measure of lightness (inverse degree of melanism). Overall, wing size increased with latitude by ∼2% per 100 km, consistent with Bergmann's rule. Forewings became more rounded and hindwings more elongated with history of colonisation, possibly reflecting selection for increased dispersal ability. Contrary to thermal melanism expectations, wing colour was lighter where larvae developed at cooler temperatures and unrelated to long-term temperature. Changes in wing spot pattern were also detected. High heterogeneity in variance among sites for all of the traits studied may reflect evolutionary time-lags and genetic drift due to colonisation of new habitats. Our study suggests that temperature-sensitive plastic responses for size and colour interact with selection for dispersal traits (wing size and shape). Whilst the plastic and evolutionary responses may in some cases act antagonistically, the rapid expansion of P. aegeria implies an overall reinforcing effect between these two mechanisms.

Adaptive colour change and background choice behaviour in peppered moth caterpillars is mediated by extraocular photoreception.

Light sensing by tissues distinct from the eye occurs in diverse animal groups, enabling circadian control and phototactic behaviour. Extraocular photoreceptors may also facilitate rapid colour change in cephalopods and lizards, but little is known about the sensory system that mediates slow colour change in arthropods. We previously reported that slow colour change in twig-mimicking caterpillars of the peppered moth (Biston betularia) is a response to achromatic and chromatic visual cues. Here we show that the perception of these cues, and the resulting phenotypic responses, does not require ocular vision. Caterpillars with completely obscured ocelli remained capable of enhancing their crypsis by changing colour and choosing to rest on colour-matching twigs. A suite of visual genes, expressed across the larval integument, likely plays a key role in the mechanism. To our knowledge, this is the first evidence that extraocular colour sensing can mediate pigment-based colour change and behaviour in an arthropod.

Climate-induced phenology shifts linked to range expansions in species with multiple reproductive cycles per year.

Advances in phenology (the annual timing of species' life-cycles) in response to climate change are generally viewed as bioindicators of climate change, but have not been considered as predictors of range expansions. Here, we show that phenology advances combine with the number of reproductive cycles per year (voltinism) to shape abundance and distribution trends in 130 species of British Lepidoptera, in response to ~0.5 °C spring-temperature warming between 1995 and 2014. Early adult emergence in warm years resulted in increased within- and between-year population growth for species with multiple reproductive cycles per year (n = 39 multivoltine species). By contrast, early emergence had neutral or negative consequences for species with a single annual reproductive cycle (n = 91 univoltine species), depending on habitat specialisation. We conclude that phenology advances facilitate polewards range expansions in species exhibiting plasticity for both phenology and voltinism, but may inhibit expansion by less flexible species.

Colour change of twig-mimicking peppered moth larvae is a continuous reaction norm that increases camouflage against avian predators.

Camouflage, and in particular background-matching, is one of the most common anti-predator strategies observed in nature. Animals can improve their match to the colour/pattern of their surroundings through background selection, and/or by plastic colour change. Colour change can occur rapidly (a few seconds), or it may be slow, taking hours to days. Many studies have explored the cues and mechanisms behind rapid colour change, but there is a considerable lack of information about slow colour change in the context of predation: the cues that initiate it, and the range of phenotypes that are produced. Here we show that peppered moth (Biston betularia) larvae respond to colour and luminance of the twigs they rest on, and exhibit a continuous reaction norm of phenotypes. When presented with a heterogeneous environment of mixed twig colours, individual larvae specialise crypsis towards one colour rather than developing an intermediate colour. Flexible colour change in this species has likely evolved in association with wind dispersal and polyphagy, which result in caterpillars settling and feeding in a diverse range of visual environments. This is the first example of visually induced slow colour change in Lepidoptera that has been objectively quantified and measured from the visual perspective of natural predators.

A high-coverage draft genome of the mycalesine butterfly Bicyclus anynana.

The mycalesine butterfly Bicyclus anynana, the "Squinting bush brown," is a model organism in the study of lepidopteran ecology, development, and evolution. Here, we present a draft genome sequence for B. anynana to serve as a genomics resource for current and future studies of this important model species. Seven libraries with insert sizes ranging from 350 bp to 20 kb were constructed using DNA from an inbred female and sequenced using both Illumina and PacBio technology; 128 Gb of raw Illumina data was filtered to 124 Gb and assembled to a final size of 475 Mb (∼×260 assembly coverage). Contigs were scaffolded using mate-pair, transcriptome, and PacBio data into 10 800 sequences with an N50 of 638 kb (longest scaffold 5 Mb). The genome is comprised of 26% repetitive elements and encodes a total of 22 642 predicted protein-coding genes. Recovery of a BUSCO set of core metazoan genes was almost complete (98%). Overall, these metrics compare well with other recently published lepidopteran genomes. We report a high-quality draft genome sequence for Bicyclus anynana. The genome assembly and annotated gene models are available at LepBase (http://ensembl.lepbase.org/index.html).

Inbreeding uncovers fundamental differences in the genetic load affecting male and female fertility in a butterfly.

Inbreeding depression is most pronounced for traits closely associated with fitness. The traditional explanation is that natural selection eliminates deleterious mutations with additive or dominant effects more effectively than recessive mutations, leading to directional dominance for traits subject to strong directional selection. Here we report the unexpected finding that, in the butterfly Bicyclus anynana, male sterility contributes disproportionately to inbreeding depression for fitness (complete sterility in about half the sons from brother-sister matings), while female fertility is insensitive to inbreeding. The contrast between the sexes for functionally equivalent traits is inconsistent with standard selection arguments, and suggests that trait-specific developmental properties and cryptic selection play crucial roles in shaping genetic architecture. There is evidence that spermatogenesis is less developmentally stable than oogenesis, though the unusually high male fertility load in B. anynana additionally suggests the operation of complex selection maintaining male sterility recessives. Analysis of the precise causes of inbreeding depression will be needed to generate a model that reliably explains variation in directional dominance and reconciles the gap between observed and expected genetic loads carried by populations. This challenging evolutionary puzzle should stimulate work on the occurrence and causes of sex differences in fertility load.

Male emergence schedule and dispersal behaviour are modified by mate availability in heterogeneous landscapes: evidence from the orange-tip butterfly.

Protandry (prior emergence of males) in insect populations is usually considered to be the result of natural selection acting directly on eclosion timing. When females are monandrous (mate once), males in high density populations benefit from early emergence in the intense scramble competition for mates. In low density populations, however, scramble competition is reduced or absent, and theoretical models predict that protandry will be less favoured. This raises the question of how males behave in heterogeneous landscapes characterized by high density core populations in a low density continuum. We hypothesized that disadvantaged late emerging males in a core population would disperse to the continuum to find mates. We tested this idea using the protandrous, monandrous, pierid butterfly Anthocharis cardamines (the orange-tip) in a core population in Cheshire, northwest England. Over a six-year period, predicted male fitness (the number of matings a male can expect during his residence time, determined by the daily ratio of virgin females to competing males) consistently declined to <1 in late season. This decline affected a large proportion (∼44%) of males in the population and was strongly associated with decreased male recapture-rates, which we attribute to dispersal to the surrounding continuum. In contrast, reanalysis of mark-release-recapture data from an isolated population in Durham, northeast England, showed that in the absence of a continuum very few males (∼3%) emerged when fitness declined to <1 in late season. Hence the existence of a low density continuum may lead to the evolution of plastic dispersal behaviour in high density core populations, maintaining late emerging males which would otherwise be eliminated by selection. This has important theoretical consequences, since a truncated male emergence curve is a key prediction in game theoretic models of emergence timing which has so far received limited support. Our results have implications for conservation, since plastic dispersal behaviour in response to imperfect emergence timing in core (source) populations could help to maintain sink populations in heterogeneous landscapes which would otherwise be driven to extinction by low mate encounter-rates (Allee effects).

Rapid spread of immigrant genomes into inbred populations.

When local populations are genetically differentiated from one another and partially inbred, as typically occurs in subdivided populations, immigrant genomes are predicted to be at a frequency-dependent fitness advantage due to heterosis (hybrid vigour) in their descendants. We tested this prediction with pedigreed laboratory populations of the butterfly Bicyclus anynana and report here on a rapid increase over five generations in the contribution of an initially rare immigrant genome to the local population gene pool. The replicated experimental design, including immigrant controls, demonstrates that the mechanism underlying immigrant genome spread is heterosis, and that the advantage to the immigrant genes is sustained over several generations. Our result suggests that effective migration rates may often be much higher than the numbers of individual migrants assumed by classical population genetics models, with implications for the persistence and evolution of metapopulations.

Industrial melanism in British peppered moths has a singular and recent mutational origin.

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 is not associated with genetic variation in canonical melanisation gene candidates.

Industrial melanism in the peppered moth (Biston betularia) is an iconic case study of ecological genetics but the molecular identity of the gene determining the difference between the typical and melanic (carbonaria) morphs is entirely unknown. We applied the candidate gene approach to look for associations between genetic polymorphisms within sixteen a priori melanisation gene candidates and the carbonaria morph. The genes were isolated and sequence characterised in B. betularia using degenerate PCR and from whole-transcriptome sequence. The list of candidates contains all the genes previously implicated in melanisation pattern differences in other insects, including aaNAT, DOPA-decarboxylase, ebony, tan, tyrosine hydroxylase, yellow and yellow2 (yellow-fa). Co-segregation of candidate gene alleles and carbonaria morph was tested in 73 offspring of a carbonaria male-typical female backcross. Surprisingly, none of the sixteen candidate genes was in close linkage with the locus controlling the carbonaria-typical polymorphism. Our study demonstrates that the 'carbonaria gene' is not a structural variant of a canonical melanisation pathway gene, neither is it a cis-regulatory element of these enzyme-coding genes. The implication is either that we have failed to characterize an unknown enzyme-coding gene in the melanisation pathway, or more likely, that the 'carbonaria gene' is a higher level trans-acting factor which regulates the spatial expression of one or more of the melanisation candidates in this study to alter the pattern of melanin production.