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1.  Molecular basis of wing coloration in a Batesian mimic butterfly, Papilio polytes 
Scientific Reports  2013;3:3184.
Batesian mimicry protects animals from predators through resemblance with distasteful models in shape, color pattern, or behavior. To elucidate the wing coloration mechanisms involved in the mimicry, we investigated chemical composition and gene expression of the pale yellow and red pigments of a swallowtail butterfly, Papilio polytes, whose females mimic the unpalatable butterfly Pachliopta aristolochiae. Using LC/MS, we showed that the pale yellow wing regions in non-mimetic females consist of kynurenine and N-β-alanyldopamine (NBAD). Moreover, qRT-PCR showed that kynurenine/NBAD biosynthetic genes were upregulated in these regions in non-mimetic females. However, these pigments were absent in mimetic females. RNA-sequencing showed that kynurenine/NBAD synthesis and Toll signaling genes were upregulated in the red spots specific to mimetic female wings. These results demonstrated that drastic changes in gene networks in the red and pale yellow regions can switch wing color patterns between non-mimetic and mimetic females of P. polytes.
PMCID: PMC3822385  PMID: 24212474
2.  Tetrachromacy in a butterfly that has eight varieties of spectral receptors 
This paper presents the first evidence of tetrachromacy among invertebrates. The Japanese yellow swallowtail butterfly, Papilio xuthus, uses colour vision when foraging. The retina of Papilio is furnished with eight varieties of spectral receptors of six classes that are the ultraviolet (UV), violet, blue (narrow-band and wide-band), green (single-peaked and double-peaked), red and broad-band classes. We investigated whether all of the spectral receptors are involved in colour vision by measuring the wavelength discrimination ability of foraging Papilio. We trained Papilio to take nectar while seeing a certain monochromatic light. We then let the trained Papilio choose between two lights of different wavelengths and determined the minimum discriminable wavelength difference Δλ. The Δλ function of Papilio has three minima at approximately 430, 480 and 560 nm, where the Δλ values approximately 1 nm. This is the smallest value found for wavelength discrimination so far, including that of humans. The profile of the Δλ function of Papilio can be best reproduced by postulating that the UV, blue (narrow-band and wide-band), green (double-peaked) and red classes are involved in foraging. Papilio colour vision is therefore tetrachromatic.
PMCID: PMC2599938  PMID: 18230593
colour vision; wavelength discrimination; spectral receptor; compound eye; ommatidium
3.  Evolutionary history of the recruitment of conserved developmental genes in association to the formation and diversification of a novel trait 
The origin and modification of novel traits are important aspects of biological diversification. Studies combining concepts and approaches of developmental genetics and evolutionary biology have uncovered many examples of the recruitment, or co-option, of genes conserved across lineages for the formation of novel, lineage-restricted traits. However, little is known about the evolutionary history of the recruitment of those genes, and of the relationship between them -for example, whether the co-option involves whole or parts of existing networks, or whether it occurs by redeployment of individual genes with de novo rewiring. We use a model novel trait, color pattern elements on butterfly wings called eyespots, to explore these questions. Eyespots have greatly diversified under natural and sexual selection, and their formation involves genetic circuitries shared across insects.
We investigated the evolutionary history of the recruitment and co-recruitment of four conserved transcription regulators to the larval wing disc region where circular pattern elements develop. The co-localization of Antennapedia, Notch, Distal-less, and Spalt with presumptive (eye)spot organizers was examined in 13 butterfly species, providing the largest comparative dataset available for the system. We found variation between families, between subfamilies, and between tribes. Phylogenetic reconstructions by parsimony and maximum likelihood methods revealed an unambiguous evolutionary history only for Antennapedia, with a resolved single origin of eyespot-associated expression, and many homoplastic events for Notch, Distal-less, and Spalt. The flexibility in the (co-)recruitment of the targeted genes includes cases where different gene combinations are associated with morphologically similar eyespots, as well as cases where identical protein combinations are associated with very different phenotypes.
The evolutionary history of gene (co-)recruitment is consistent with both divergence from a recruited putative ancestral network, and with independent co-option of individual genes. The diversity in the combinations of genes expressed in association with eyespot formation does not parallel diversity in characteristics of the adult phenotype. We discuss these results in the context of inferring homology. Our study underscores the importance of widening the representation of phylogenetic, morphological, and genetic diversity in order to establish general principles about the mechanisms behind the evolution of novel traits.
PMCID: PMC3361465  PMID: 22335999
4.  Simultaneous brightness contrast of foraging Papilio butterflies 
This study focuses on the sense of brightness in the foraging Japanese yellow swallowtail butterfly, Papilio xuthus. We presented two red discs of different intensity on a grey background to butterflies, and trained them to select one of the discs. They were successfully trained to select either a high intensity or a low intensity disc. The trained butterflies were tested on their ability to perceive brightness in two different protocols: (i) two orange discs of different intensity presented on the same intensity grey background and (ii) two orange discs of the same intensity separately presented on a grey background that was either higher or lower in intensity than the training background. The butterflies trained to high intensity red selected the orange disc of high intensity in protocol 1, and the disc on the background of low intensity grey in protocol 2. We obtained similar results in another set of experiments with purple discs instead of orange discs. The choices of the butterflies trained to low intensity red were opposite to those just described. Taken together, we conclude that Papilio has the ability to learn brightness and darkness of targets independent of colour, and that they have the so-called simultaneous brightness contrast.
PMCID: PMC3311899  PMID: 22179808
vision; insect; compound eye; neuroethology
5.  Comparative insights into questions of lepidopteran wing pattern homology 
Butterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occuring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-β signaling pathways as putative morphogens for eyespot development.
Saturniid moth and nymphalid butterfly eyespots with concentric rings of color express at least two transcription factors, Distal-less and Engrailed, in the center of the future pattern. Nymphalid eyespots centers also express the ligand Wingless and an activated signal transducer, a phosphorylated Smad protein, but neither these proteins nor the previous two proteins are found in pierid spot centers, which consist of a single patch of color. Both butterfly wing patterns, however, express a third transcription factor, Spalt, a portion of whose expression domain maps to the black scales on the adult wing. Wounding a nymphalid wing, on the other hand, leads to upregulation of Distal-less, engrailed and spalt in subsets of cells around the wounding site, mimicking concentric eyespot development.
Wingless and TGF-β ligands are both candidate morphogens involved in nymphalid butterfly eyespot formation. These eyespots, as well as saturniid moth eyespots with concentric circles, share two genes that are associated with the differentiation of the signaling cells in nymphalid eyespots. This commonality suggests that they may be produced via the same developmental mechanism despite their non-homologous location. By contrast, pierid butterfly spots of a single color share some of the same genes but appear to be produced by a different mechanism. Eyespots with concentric rings may have co-opted a wound healing genetic network during their evolution.
PMCID: PMC1654149  PMID: 17090321
6.  Deimatic Display in the European Swallowtail Butterfly as a Secondary Defence against Attacks from Great Tits 
PLoS ONE  2012;7(10):e47092.
Many animals reduce the risk of being attacked by a predator through crypsis, masquerade or, alternatively, by advertising unprofitability by means of aposematic signalling. Behavioural attributes in prey employed after discovery, however, signify the importance of also having an effective secondary defence if a predator uncovers, or is immune to, the prey’s primary defence. In butterflies, as in most animals, secondary defence generally consists of escape flights. However, some butterfly species have evolved other means of secondary defence such as deimatic displays/startle displays. The European swallowtail, Papilio machaon, employs what appears to be a startle display by exposing its brightly coloured dorsal wing surface upon disturbance and, if the disturbance continues, by intermittently protracting and relaxing its wing muscles generating a jerky motion of the wings. This display appears directed towards predators but whether it is effective in intimidating predators so that they refrain from attacks has never been tested experimentally.
Methodology/Principal Findings
In this study we staged encounters between a passerine predator, the great tit, Parus major, and live and dead swallowtail butterflies in a two-choice experiment. Results showed that the dead butterfly was virtually always attacked before the live butterfly, and that it took four times longer before a bird attacked the live butterfly. When the live butterfly was approached by a bird this generally elicited the butterfly’s startle display, which usually caused the approaching bird to flee. We also performed a palatability test of the butterflies and results show that the great tits seemed to find them palatable.
We conclude that the swallowtail’s startle display of conspicuous coloration and jerky movements is an efficient secondary defence against small passerines. We also discuss under what conditions predator-prey systems are likely to aid the evolution of deimatic behaviours in harmless and palatable prey.
PMCID: PMC3466272  PMID: 23056590
7.  Genomic Hotspots for Adaptation: The Population Genetics of Müllerian Mimicry in Heliconius erato 
PLoS Genetics  2010;6(2):e1000796.
Wing pattern evolution in Heliconius butterflies provides some of the most striking examples of adaptation by natural selection. The genes controlling pattern variation are classic examples of Mendelian loci of large effect, where allelic variation causes large and discrete phenotypic changes and is responsible for both convergent and highly divergent wing pattern evolution across the genus. We characterize nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium (LD), and candidate gene expression patterns across two unlinked genomic intervals that control yellow and red wing pattern variation among mimetic forms of Heliconius erato. Despite very strong natural selection on color pattern, we see neither a strong reduction in genetic diversity nor evidence for extended LD across either patterning interval. This observation highlights the extent that recombination can erase the signature of selection in natural populations and is consistent with the hypothesis that either the adaptive radiation or the alleles controlling it are quite old. However, across both patterning intervals we identified SNPs clustered in several coding regions that were strongly associated with color pattern phenotype. Interestingly, coding regions with associated SNPs were widely separated, suggesting that color pattern alleles may be composed of multiple functional sites, conforming to previous descriptions of these loci as “supergenes.” Examination of gene expression levels of genes flanking these regions in both H. erato and its co-mimic, H. melpomene, implicate a gene with high sequence similarity to a kinesin as playing a key role in modulating pattern and provides convincing evidence for parallel changes in gene regulation across co-mimetic lineages. The complex genetic architecture at these color pattern loci stands in marked contrast to the single casual mutations often identified in genetic studies of adaptation, but may be more indicative of the type of genetic changes responsible for much of the adaptive variation found in natural populations.
Author Summary
Identifying the genetic changes responsible for beneficial variation is essential for understanding how organisms adapt. Here, we use a combination of mapping, population genetic analysis, and gene expression studies to identify the genomic regions responsible for phenotypic evolution in the Neotropical butterfly Heliconius erato. H. erato, together with its co-mimic H. melpomene, have undergone parallel and concordant radiations in their warningly colored wing patterns across Central and South America. The “genes” underlying the H. erato color pattern radiation are classic examples of Mendelian loci of large effect and are under strong natural selection. Nonetheless, we do not see a clear molecular signal of recent natural selection, suggesting that the H. erato color pattern radiation, or the alleles that underlie it, may be quite old. Moreover, rather than being single locus, the genetic patterns suggest that multiple, widely dispersed loci may underlie pattern variation in H. erato. One of these loci, a kinesin gene, shows parallel expression differences between races during wing pattern formation in both H. erato and H. melpomene, suggesting that it plays an important role in pattern variation. High rates of recombination within naturally occurring H. erato hybrid zones mean that finer genetic dissection will allow us to localize causative sites and better understand the history and molecular basis of this extraordinary adaptive radiation.
PMCID: PMC2816678  PMID: 20140239
8.  Aposematism and crypsis combined as a result of distance dependence: functional versatility of the colour pattern in the swallowtail butterfly larva 
The idea that an aposematic prey combines crypsis at a distance with conspicuousness close up was tested in an experiment using human subjects. We estimated detectability of the aposematic larva of the swallowtail butterfly, Papilio machaon, in two habitats, by presenting, on a touch screen, photographs taken at four different distances and measuring the time elapsed to discovery. The detectability of larvae in these images was compared with images that were manipulated, using existing colours either to increase or decrease conspicuousness. Detection time increased with distance for all colourations. However, at the closest distance, detection time was longer for the larvae manipulated to be more cryptic than for the natural and more conspicuous forms. This indicates that the natural colouration is not maximally cryptic at a short distance. Further, smaller increments in distance were needed to increase detection time for the natural than for the conspicuous larva. This indicates that the natural colouration is not maximally conspicuous at longer distances. Taken together, we present the first empirical support for the idea that some colour patterns may combine warning colouration at a close range with crypsis at a longer range. The implications of this result for the evolution of aposematism are discussed.
PMCID: PMC1560331  PMID: 16006332
aposematism; crypsis; conspicuousness; predation; distance; background
9.  Genome-Wide Characterization of Adaptation and Speciation in Tiger Swallowtail Butterflies Using De Novo Transcriptome Assemblies 
Genome Biology and Evolution  2013;5(6):1233-1245.
Hybrid speciation appears to be rare in animals, yet characterization of possible examples offers to shed light on the genomic consequences of this unique phenomenon, as well as more general processes such as the role of adaptation in speciation. Here, we first generate transcriptome assemblies for a putative hybrid butterfly species, Papilio appalachiensis, its parental species, P. glaucus and P. canadensis, and an outgroup, P. polytes. Then, we use these data to infer genome-wide patterns of introgression and genomic mosaicism using both phylogenetic and population genetic approaches. Our results reveal that there is little genetic divergence among all three of the focal species, but the subset of gene trees that strongly support a specific tree topology suggest widespread sharing of genetic variation between P. appalachiensis and both parental species, likely as a result of hybrid speciation. We also find evidence for substantial shared genetic variation between P. glaucus and P. canadensis, which may be due to gene flow or ancestral variation. Consistent with previous work, we show that P. applachiensis is more similar to P. canadensis at Z-linked genes and more similar to P. glaucus at mitochondrial genes. We also identify a variety of targets of adaptive evolution, which appear to be enriched for traits that are likely to be important in the evolution of this butterfly system, such as pigmentation, hormone sensitivity, developmental processes, and cuticle formation. Overall, our results provide a genome-wide portrait of divergence and introgression associated with adaptation and speciation in an iconic butterfly radiation.
PMCID: PMC3698933  PMID: 23737327
transcriptome; adaptation; hybrid speciation; introgression; Papilio
10.  Catalogue of epidermal genes: Genes expressed in the epidermis during larval molt of the silkworm Bombyx mori 
BMC Genomics  2008;9:396.
The insect cuticle is composed of various proteins and formed during the molt under hormonal regulation, although its precise composition and formation mechanism are largely unknown. The exhaustive catalogue of genes expressed in epidermis at the molt constitutes a massive amount of information from which to draw a complete picture of the molt and cuticle formation in insects. Therefore, we have catalogued a library of full-length cDNAs (designated epM) from epidermal cells during the last larval molt of Bombyx mori.
Of the 10,368 sequences in the library, we isolated 6,653 usable expressed sequence tags (ESTs), which were categorized into 1,451 nonredundant gene clusters. Seventy-one clusters were considered to be isoforms or premature forms of other clusters. Therefore, we have identified 1,380 putative genes. Of the 6,653 expressed sequences, 48% were derived from 92 cuticular protein genes (RR-1, 24; RR-2, 17; glycine-rich, 29; other classes, 22). A comparison of epM with another epidermal EST data set, epV3 (feeding stage: fifth instar, day 3), showed marked differences in cuticular protein gene. Various types of cuticular proteins are expressed in epM but virtually only RR-1 proteins were expressed in epV3. Cuticular protein genes expressed specifically in epidermis, with several types of expression patterns during the molt, suggest different types of responses to the ecdysteroid pulse. Compared with other Bombyx EST libraries, 13 genes were preferentially included in epM data set. We isolated 290 genes for proteins other than cuticular proteins, whose amino acid sequences retain putative signal peptides, suggesting that they play some role in cuticle formation or in other molting events. Several gene groups were also included in this data set: hormone metabolism, P450, modifier of cuticular protein structure, small-ligand-binding protein, transcription factor, and pigmentation genes.
We have identified 1,380 genes in epM data set and 13 preferentially expressed genes in epidermis at the molt. The comparison of the epM and other EST libraries clarified the totally different gene expression patterns in epidermis between the molting and feeding stages and many novel tissue- and stage-specifically expressed epidermal genes. These data should further our understanding of cuticle formation and the insect molt.
PMCID: PMC2542385  PMID: 18721459
11.  Population-level transcriptome sequencing of nonmodel organisms Erynnis propertius and Papilio zelicaon 
BMC Genomics  2010;11:310.
Several recent studies have demonstrated the use of Roche 454 sequencing technology for de novo transcriptome analysis. Low error rates and high coverage also allow for effective SNP discovery and genetic diversity estimates. However, genetically diverse datasets, such as those sourced from natural populations, pose challenges for assembly programs and subsequent analysis. Further, estimating the effectiveness of transcript discovery using Roche 454 transcriptome data is still a difficult task.
Using the Roche 454 FLX Titanium platform, we sequenced and assembled larval transcriptomes for two butterfly species: the Propertius duskywing, Erynnis propertius (Lepidoptera: Hesperiidae) and the Anise swallowtail, Papilio zelicaon (Lepidoptera: Papilionidae). The Expressed Sequence Tags (ESTs) generated represent a diverse sample drawn from multiple populations, developmental stages, and stress treatments.
Despite this diversity, > 95% of the ESTs assembled into long (> 714 bp on average) and highly covered (> 9.6× on average) contigs. To estimate the effectiveness of transcript discovery, we compared the number of bases in the hit region of unigenes (contigs and singletons) to the length of the best match silkworm (Bombyx mori) protein--this "ortholog hit ratio" gives a close estimate on the amount of the transcript discovered relative to a model lepidopteran genome. For each species, we tested two assembly programs and two parameter sets; although CAP3 is commonly used for such data, the assemblies produced by Celera Assembler with modified parameters were chosen over those produced by CAP3 based on contig and singleton counts as well as ortholog hit ratio analysis. In the final assemblies, 1,413 E. propertius and 1,940 P. zelicaon unigenes had a ratio > 0.8; 2,866 E. propertius and 4,015 P. zelicaon unigenes had a ratio > 0.5.
Ultimately, these assemblies and SNP data will be used to generate microarrays for ecoinformatics examining climate change tolerance of different natural populations. These studies will benefit from high quality assemblies with few singletons (less than 26% of bases for each assembled transcriptome are present in unassembled singleton ESTs) and effective transcript discovery (over 6,500 of our putative orthologs cover at least 50% of the corresponding model silkworm gene).
PMCID: PMC2887415  PMID: 20478048
12.  Colour pattern specification in the Mocker swallowtail Papilio dardanus: the transcription factor invected is a candidate for the mimicry locus H 
The swallowtail butterfly, Papilio dardanus, is an iconic example of a polymorphic Batesian mimic. The expression of various female-limited colour forms is thought to be controlled by a single autosomal locus, termed H, whose function in determining the wing pattern remains elusive. As a step towards the physical mapping of H, we established a set of 272 polymorphic amplified fragment length polymorphism (AFLP) markers (EcoRI-MseI). Segregation patterns in a ‘female-informative’ brood (exploiting the absence of crossing over in female Lepidoptera) mapped these AFLPs to 30 linkage groups (putative chromosomes). The difference between the hippocoon and cenea female forms segregating in this family resides on a single one of these linkage groups, defined by 14 AFLPs. In a ‘male-informative’ cross (markers segregating within a linkage group), a pair of AFLPs co-segregated closely with the two female forms, except in four recombinants out of 19 female offspring. Linkage with these AFLP markers using four further female-informative families demonstrated that the genetic factor determining other morphs (poultoni, lamborni and trimeni) also maps to this same linkage group. The candidate gene invected, obtained in a screen for co-segregation of developmental genes with the colour forms, resides in a 13.9 cM interval flanked by the two AFLP markers. In the male-informative family invected co-segregated perfectly with the hippocoon/cenea factor, despite the four crossovers with the AFLPs. These findings make invected, and possibly its closely linked paralogue engrailed, strong candidates for H. This is supported by their known role in eyespot specification in nymphalid butterfly wings.
PMCID: PMC2602692  PMID: 18285283
evolutionary genetics; mimicry; phenotype–genotype association; engrailed; candidate genes; amplified fragment length polymorphism
13.  Mimetic butterflies support Wallace's model of sexual dimorphism 
Theoretical and empirical observations generally support Darwin's view that sexual dimorphism evolves due to sexual selection on, and deviation in, exaggerated male traits. Wallace presented a radical alternative, which is largely untested, that sexual dimorphism results from naturally selected deviation in protective female coloration. This leads to the prediction that deviation in female rather than male phenotype causes sexual dimorphism. Here I test Wallace's model of sexual dimorphism by tracing the evolutionary history of Batesian mimicry—an example of naturally selected protective coloration—on a molecular phylogeny of Papilio butterflies. I show that sexual dimorphism in Papilio is significantly correlated with both female-limited Batesian mimicry, where females are mimetic and males are non-mimetic, and with the deviation of female wing colour patterns from the ancestral patterns conserved in males. Thus, Wallace's model largely explains sexual dimorphism in Papilio. This finding, along with indirect support from recent studies on birds and lizards, suggests that Wallace's model may be more widely useful in explaining sexual dimorphism. These results also highlight the contribution of naturally selected female traits in driving phenotypic divergence between species, instead of merely facilitating the divergence in male sexual traits as described by Darwin's model.
PMCID: PMC2602815  PMID: 18426753
Batesian mimicry; polymorphism; female-limited mimicry; directional selection; stabilizing sexual selection; convergence
14.  Convergent evolution of neuroendocrine control of phenotypic plasticity in pupal colour in butterflies 
Phenotypic plasticity in pupal colour occurs in three families of butterflies (the Nymphalidae, Papilionidae and Pieridae), typically in species whose pupation sites vary unpredictably in colour. In all species studied to date, larvae ready for pupation respond to environmental cues associated with the colour of their pupation sites and moult into cryptic light (yellow–green) or dark (brown–black) pupae. In nymphalids and pierids, pupal colour is controlled by a neuroendocrine factor, pupal melanization-reducing factor (PMRF), the release of which inhibits the melanization of the pupal cuticle resulting in light pupae. In contrast, the neuroendocrine factor controlling pupal colour in papilionid butterflies results in the production of brown pupae. PMRF was extracted from the ventral nerve chains of the peacock butterfly Inachis io (Nymphalidae) and black swallowtail butterfly Papilio polyxenes (Papilionidae). When injected into pre-pupae, the extracts resulted in yellow pupae in I. io but brown pupae in P. polyxenes. These results suggest that the same neuroendocrine factor controls the plasticity in pupal colour, but that plasticity in pupal colour in these species has evolved independently (convergently).
PMCID: PMC1690474
15.  Disruptive coloration provides camouflage independent of background matching 
Natural selection shapes the evolution of anti-predator defences, such as camouflage. It is currently contentious whether crypsis and disruptive coloration are alternative mechanisms of camouflage or whether they are interrelated anti-predator defences. Disruptively coloured prey is characterized by highly contrasting patterns to conceal the body shape, whereas cryptic prey minimizes the contrasts to background. Determining bird predation of artificial moths, we found that moths which were dissimilar from the background but sported disruptive patterns on the edge of their wings survived better in heterogeneous habitats than did moths with the same patterns inside of the wings and better than cryptic moths. Despite lower contrasts to background, crypsis did not provide fitness benefits over disruptive coloration on the body outline. We conclude that disruptive coloration on the edge camouflages its bearer independent of background matching. We suggest that this result is explainable because disruptive coloration is effective by exploiting predators' cognitive mechanisms of prey recognition and not their sensory mechanisms of signal detection. Relative to disruptive patterns on the body outline, disruptive markings on the body interior are less effective. Camouflage owing to disruptive coloration on the body interior is background-specific and is as effective as crypsis in heterogeneous habitats. Hence, we hypothesize that two proximate mechanisms explain the diversity of visual anti-predator defences. First, disruptive coloration on the body outline provides camouflage independent of the background. Second, background matching and disruptive coloration on the body interior provide camouflage, but their protection is background-specific.
PMCID: PMC1634905  PMID: 16959631
crypsis; natural selection; anti-predator defences; colour vision; prey recognition
16.  A model for colour pattern formation in the butterfly wing of Papilio dardanus. 
The butterfly Papilio dardanus is well known for the spectacular phenotypic polymorphism in the female of the species. We show that numerical simulations of a reaction diffusion model on a geometrically accurate wing domain produce spatial patterns that are consistent with many of those observed on the butterfly. Our results suggest that the wing coloration is due to a simple underlying stripe-like pattern of some pigment-inducing morphogen. We focus on the effect of key factors such as parameter values for mode selection, threshold values which determine colour, wing shape and boundary conditions. The generality of our approach should allow us to investigate other butterfly species. The relationship between these key factors and gene activities is discussed in the context of recent biological advances.
PMCID: PMC1690625  PMID: 10853726
17.  Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development 
PLoS ONE  2012;7(12):e51087.
Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.
PMCID: PMC3515442  PMID: 23227236
18.  Sex Chromosome Mosaicism and Hybrid Speciation among Tiger Swallowtail Butterflies 
PLoS Genetics  2011;7(9):e1002274.
Hybrid speciation, or the formation of a daughter species due to interbreeding between two parental species, is a potentially important means of diversification, because it generates new forms from existing variation. However, factors responsible for the origin and maintenance of hybrid species are largely unknown. Here we show that the North American butterfly Papilio appalachiensis is a hybrid species, with genomic admixture from Papilio glaucus and Papilio canadensis. Papilio appalachiensis has a mosaic phenotype, which is hypothesized to be the result of combining sex-linked traits from P. glaucus and P. canadensis. We show that P. appalachiensis' Z-linked genes associated with a cooler thermal habitat were inherited from P. canadensis, whereas its W-linked mimicry and mitochondrial DNA were inherited from P. glaucus. Furthermore, genome-wide AFLP markers showed nearly equal contributions from each parental species in the origin of P. appalachiensis, indicating that it formed from a burst of hybridization between the parental species, with little subsequent backcrossing. However, analyses of genetic differentiation, clustering, and polymorphism based on molecular data also showed that P. appalachiensis is genetically distinct from both parental species. Population genetic simulations revealed P. appalachiensis to be much younger than the parental species, with unidirectional gene flow from P. glaucus and P. canadensis into P. appalachiensis. Finally, phylogenetic analyses, combined with ancestral state reconstruction, showed that the two traits that define P. appalachiensis' mosaic phenotype, obligatory pupal diapause and mimicry, evolved uniquely in P. canadensis and P. glaucus, respectively, and were then recombined through hybridization to form P. appalachiensis. These results suggest that natural selection and sex-linked traits may have played an important role in the origin and maintenance of P. appalachiensis as a hybrid species. In particular, ecological barriers associated with a steep thermal cline appear to maintain the distinct, mosaic genome of P. appalachiensis despite contact and occasional hybridization with both parental species.
Author Summary
Hybridization between two species may sometimes lead to the formation of a third species. While this phenomenon is of interest because it can generate new species from existing variation, it is rare in animals. Therefore, the factors responsible for the origin and maintenance of hybrid species are largely unknown. Here we show that the butterfly Papilio appalachiensis appears to be a hybrid species, which received nearly equal contributions to its genome from P. glaucus and P. canadensis. Two sets of traits define P. appalachiensis' hybrid phenotype: like P. canadensis it inhabits a cold habitat and has a single generation every year, and like P. glaucus it mimics a toxic butterfly and its females are dimorphic. The genes responsible for these traits are on two different sex chromosomes. Our genetic data show that P. appalachiensis inherited the sex chromosome associated with the cold habitat from P. canadensis, whereas it inherited the sex chromosome associated with mimicry and dimorphism from P. glaucus. Historical climate change may have facilitated the origin of P. appalachiensis, which our data suggest coincided with the last interglacial period in North America. This example shows the importance of sex-linked traits and a potential role for natural selection in the origin and maintenance of hybrid species.
PMCID: PMC3169544  PMID: 21931567
19.  A Simulation Study of Mutations in the Genetic Regulatory Hierarchy for Butterfly Eyespot Focus Determination 
Bio Systems  2008;93(3):250-255.
The color patterns on the wings of butterflies have been an important model system in evolutionary developmental biology. A recent computational model tested genetic regulatory hierarchies hypothesized to underlie the formation of butterfly eyespot foci (Evans and Marcus, 2006). The computational model demonstrated that one proposed hierarchy was incapable of reproducing the known patterns of gene expression associated with eyespot focus determination in wild-type butterflies, but that two slightly modified alternative hierarchies were capable of reproducing all of the known gene expressions patterns. Here we extend the computational models previously implemented in Delphi 2.0 to two mutants derived from the squinting bush brown butterfly (Bicyclus anynana). These two mutants, comet and Cyclops, have aberrantly shaped eyespot foci that are produced by different mechanisms. The comet mutation appears to produce a modified interaction between the wing margin and the eyespot focus that results in a series of comet-shaped eyespot foci. The Cyclops mutation causes the failure of wing vein formation between two adjacent wing-cells and the fusion of two adjacent eyespot foci to form a single large elongated focus in their place. The computational approach to modeling pattern formation in these mutants allows us to make predictions about patterns of gene expression, which are largely unstudied in butterfly mutants. It also suggests a critical experiment that will allow us to distinguish between two hypothesized genetic regulatory hierarchies that may underlie all butterfly eyespot foci.
PMCID: PMC2553004  PMID: 18586070
butterfly eyespot; genetic regulatory hierarchy; computational models; Bicyclus anynana; wing patterns
20.  A new Hermeuptychia (Lepidoptera, Nymphalidae, Satyrinae) is sympatric and synchronic with H. sosybius in southeast US coastal plains, while another new Hermeuptychia species – not hermes – inhabits south Texas and northeast Mexico 
ZooKeys  2014;43-91.
Hermeuptychia intricata Grishin, sp. n. is described from the Brazos Bend State Park in Texas, United States, where it flies synchronously with Hermeuptychia sosybius (Fabricius, 1793). The two species differ strongly in both male and female genitalia and exhibit 3.5% difference in the COI barcode sequence of mitochondrial DNA. Setting such significant genitalic and genotypic differences aside, we were not able to find reliable wing pattern characters to tell a difference between the two species. This superficial similarity may explain why H. intricata, only distantly related to H. sosybius, has remained unnoticed until now, despite being widely distributed in the coastal plains from South Carolina to Texas, USA (and possibly to Costa Rica). Obscuring the presence of a cryptic species even further, wing patterns are variable in both butterflies and ventral eyespots vary from large to almost absent. To avoid confusion with the new species, neotype for Papilio sosybius Fabricius, 1793, a common butterfly that occurs across northeast US, is designated from Savannah, Georgia, USA. It secures the universally accepted traditional usage of this name. Furthermore, we find that DNA barcodes of Hermeuptychia specimens from the US, even those from extreme south Texas, are at least 4% different from those of H. hermes (Fabricius, 1775)—type locality Brazil: Rio de Janeiro—and suggest that the name H. hermes should not be used for USA populations, but rather reserved for the South American species. This conclusion is further supported by comparison of male genitalia. However, facies, genitalia and 2.1% different DNA barcodes set Hermeuptychia populations in the lower Rio Grande Valley of Texas apart from H. sosybius. These southern populations, also found in northeastern Mexico, are described here as Hermeuptychia hermybius Grishin, sp. n. (type locality Texas: Cameron County). While being phylogenetically closer to H. sosybius than to any other Hermeuptychia species, H. hermybius can usually be recognized by wing patterns, such as the size of eyespots and the shape of brown lines on hindwing. “Intricate Satyr” and “South Texas Satyr” are proposed as the English names for H. intricata and H. hermybius, respectively.
PMCID: PMC3935228  PMID: 24574857
Biodiversity; cryptic species; DNA barcodes; neotropical; satyr; Hermeuptychia gisella; Hermeuptychia cucullina; Hermeuptychia sosybius kappeli; female genitalia
21.  Does predation maintain eyespot plasticity in Bicyclus anynana? 
The butterfly Bicyclus anynana exhibits phenotypic plasticity involving the wet-season phenotype, which possesses marginal eyespots on the ventral surface of the wings, and the dry-season form, which lacks these eyespots. We examined the adaptive value of phenotypic plasticity of B. anynana in relation to the defence mechanisms of crypsis and deflection. We assessed the visibility differences between spotless and spotted butterflies against backgrounds of brown (dry season) or green (wet season) leaves. Spotless butterflies were highly cryptic and less predated by adult bird predators than were spotted ones when presented against brown leaf litter. However, the advantage of crypsis disappeared in the wet-season habitat as both forms were equally visible. In later experiments, naive birds presented with resting butterflies in the wet-season habitat tended to learn more rapidly to capture spotless butterflies, suggesting a slight selective advantage of possessing eyespots. Moreover, marginal eyespots increased significantly the escape probability of butterflies that were attacked by naive birds compared to those attacked by adult birds, although there were no differences in prey capture success within naive predators. Our results show that natural selection acts against eyespots in the dry season, favouring crypsis, whereas in the wet season it may favour eyespots as deflective patterns.
PMCID: PMC1691594  PMID: 15058439
22.  Adaptive Variation in Beach Mice Produced by Two Interacting Pigmentation Genes 
PLoS Biology  2007;5(9):e219.
Little is known about the genetic basis of ecologically important morphological variation such as the diverse color patterns of mammals. Here we identify genetic changes contributing to an adaptive difference in color pattern between two subspecies of oldfield mice (Peromyscus polionotus). One mainland subspecies has a cryptic dark brown dorsal coat, while a younger beach-dwelling subspecies has a lighter coat produced by natural selection for camouflage on pale coastal sand dunes. Using genome-wide linkage mapping, we identified three chromosomal regions (two of major and one of minor effect) associated with differences in pigmentation traits. Two candidate genes, the melanocortin-1 receptor (Mc1r) and its antagonist, the Agouti signaling protein (Agouti), map to independent regions that together are responsible for most of the difference in pigmentation between subspecies. A derived mutation in the coding region of Mc1r, rather than change in its expression level, contributes to light pigmentation. Conversely, beach mice have a derived increase in Agouti mRNA expression but no changes in protein sequence. These two genes also interact epistatically: the phenotypic effects of Mc1r are visible only in genetic backgrounds containing the derived Agouti allele. These results demonstrate that cryptic coloration can be based largely on a few interacting genes of major effect.
Author Summary
The tremendous amount of variation in color patterns among organisms helps individuals survive and reproduce in the wild, yet we know surprisingly little about the genes that produce these adaptive patterns. Here we used a genomic analysis to uncover the molecular basis of a pale color pattern that camouflages beach mice inhabiting the sandy dunes of Florida's coast from predators. We identified two pigmentation genes, the melanocortin-1 receptor (Mc1r) and its ligand, the agouti signaling protein (Agouti), which together produce a light color pattern. We show that this light pigmentation results partly from a single amino acid mutation in Mc1r, which reduces the activity of the receptor but does not affect the gene's expression level, and partly from the derived Agouti allele, which shows no change in protein sequence but does exhibit an increase in mRNA expression. We also show that these two genes do not act additively to produce pale color; rather, the derived Agouti allele must be present to see any effect of Mc1r on pigmentation. Thus, the light color pattern of beach mice largely results from the physical interaction between a structural change in a receptor (reducing Mc1r activity) and a regulatory change in the receptor's antagonist (increasing Agouti expression).
Species of oldfield mice have coat colors adapted for their environment. By using genome-wide linkage mapping, the authors show that three chromosomal regions are associated with differences in pigmentation traits.
PMCID: PMC1945039  PMID: 17696646
23.  Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation 
Heredity  2012;108(6):594-601.
Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration.
PMCID: PMC3356807  PMID: 22234245
evo-devo; pigmentation; lepidoptera; melanogenesis; PLP-dependent enzyme; black
24.  A wing expressed sequence tag resource for Bicyclus anynana butterflies, an evo-devo model 
BMC Genomics  2006;7:130.
Butterfly wing color patterns are a key model for integrating evolutionary developmental biology and the study of adaptive morphological evolution. Yet, despite the biological, economical and educational value of butterflies they are still relatively under-represented in terms of available genomic resources. Here, we describe an Expression Sequence Tag (EST) project for Bicyclus anynana that has identified the largest available collection to date of expressed genes for any butterfly.
By targeting cDNAs from developing wings at the stages when pattern is specified, we biased gene discovery towards genes potentially involved in pattern formation. Assembly of 9,903 ESTs from a subtracted library allowed us to identify 4,251 genes of which 2,461 were annotated based on BLAST analyses against relevant gene collections. Gene prediction software identified 2,202 peptides, of which 215 longer than 100 amino acids had no homology to any known proteins and, thus, potentially represent novel or highly diverged butterfly genes. We combined gene and Single Nucleotide Polymorphism (SNP) identification by constructing cDNA libraries from pools of outbred individuals, and by sequencing clones from the 3' end to maximize alignment depth. Alignments of multi-member contigs allowed us to identify over 14,000 putative SNPs, with 316 genes having at least one high confidence double-hit SNP. We furthermore identified 320 microsatellites in transcribed genes that can potentially be used as genetic markers.
Our project was designed to combine gene and sequence polymorphism discovery and has generated the largest gene collection available for any butterfly and many potential markers in expressed genes. These resources will be invaluable for exploring the potential of B. anynana in particular, and butterflies in general, as models in ecological, evolutionary, and developmental genetics.
PMCID: PMC1534037  PMID: 16737530
25.  Once a Batesian mimic, not always a Batesian mimic: mimic reverts back to ancestral phenotype when the model is absent 
Batesian mimics gain protection from predation through the evolution of physical similarities to a model species that possesses anti-predator defences. This protection should not be effective in the absence of the model since the predator does not identify the mimic as potentially dangerous and both the model and the mimic are highly conspicuous. Thus, Batesian mimics should probably encounter strong predation pressure outside the geographical range of the model species. There are several documented examples of Batesian mimics occurring in locations without their models, but the evolutionary responses remain largely unidentified. A mimetic species has four alternative evolutionary responses to the loss of model presence. If predation is weak, it could maintain its mimetic signal. If predation is intense, it is widely presumed the mimic will go extinct. However, the mimic could also evolve a new colour pattern to mimic another model species or it could revert back to its ancestral, less conspicuous phenotype. We used molecular phylogenetic approaches to reconstruct and test the evolution of mimicry in the North American admiral butterflies (Limenitis: Nymphalidae). We confirmed that the more cryptic white-banded form is the ancestral phenotype of North American admiral butterflies. However, one species, Limenitis arthemis, evolved the black pipevine swallowtail mimetic form but later reverted to the white-banded more cryptic ancestral form. This character reversion is strongly correlated with the geographical absence of the model species and its host plant, but not the host plant distribution of L. arthemis. Our results support the prediction that a Batesian mimic does not persist in locations without its model, but it does not go extinct either. The mimic can revert back to its ancestral, less conspicuous form and persist.
PMCID: PMC2602694  PMID: 18285285
character evolution; Lepidoptera; parametric bootstrap; wing pattern evolution

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