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

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 commonanti-predator strategies observed in nature. Animals can improve their match to thecolour/pattern of their surroundings through background selection, and/or by plasticcolour 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 behindrapid colour change, but there is a considerable lack of information about slow colourchange in the context of predation: the cues that initiate it, and the range of phenotypesthat are produced. Here we show that peppered moth (Biston betularia) larvae respondto colour and luminance of the twigs they rest on, and exhibit a continuous reactionnorm of phenotypes. When presented with a heterogeneous environment of mixed twigcolours, individual larvae specialise crypsis towards one colour rather than developingan intermediate colour. Flexible colour change in this species has likely evolved inassociation with wind dispersal and polyphagy, which result in caterpillars settling andfeeding in a diverse range of visual environments. This is the first example of visuallyinduced slow colour change in Lepidoptera that has been objectively quantified andmeasured from the visual perspective of natural predators

The genome sequence of the rosy footman, Miltochrista miniata (Forster, 1771)

We present a genome assembly from an individual female Miltochrista miniata (the Rosy Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 1057.0 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.93 kilobases in length. Gene annotation of this assembly on Ensembl identified 23,879 protein coding genes

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

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

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, intra-locus genetic variation and inter-locus similarity within these data. Here we present a comparative analysis of assembly algorithms representing overlap-layout-consensus, repeat graph and de Brujn graph methods. We examine how post-processing 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 non-coding diversity between redundant sequences. We also document a link between an excess of non-synonymous polymorphism and haplotigs that are unresolved by assembly or post-assembly algorithms. Finally, we discuss how this phenomenon may have relevance for the usage of noisy long-read genome assemblies in comparative genomics

A high-coverage draft genome of the mycalesine butterfly <i>Bicyclus anynana</i>

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)

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).Peer reviewe