Acute and Long-Term Consequences of Co-opted doublesex on the Development of Mimetic Butterfly Color Patterns.

Novel phenotypes are increasingly recognized to have evolved by co-option of conserved genes into new developmental contexts, yet the process by which co-opted genes modify existing developmental programs remains obscure. Here, we provide insight into this process by characterizing the role of co-opted doublesex in butterfly wing color pattern development. dsx is the master regulator of insect sex differentiation but has been co-opted to control the switch between discrete nonmimetic and mimetic patterns in Papilio alphenor and its relatives through the evolution of novel mimetic alleles. We found dynamic spatial and temporal expression pattern differences between mimetic and nonmimetic butterflies throughout wing development. A mimetic color pattern program is switched on by a pulse of dsx expression in early pupal development that causes acute and long-term differential gene expression, particularly in Wnt and Hedgehog signaling pathways. RNAi suggested opposing, novel roles for these pathways in mimetic pattern development. Importantly, Dsx co-option caused Engrailed, a primary target of Hedgehog signaling, to gain a novel expression domain early in pupal wing development that is propagated through mid-pupal development to specify novel mimetic patterns despite becoming decoupled from Dsx expression itself. Altogether, our findings provide multiple views into how co-opted genes can both cause and elicit changes to conserved networks and pathways to result in development of novel, adaptive phenotypes.

Divergent expression of aristaless1 and aristaless2 during embryonic appendage and pupal wing development in butterflies.

BACKGROUND: Gene duplication events are critical for the evolution of new gene functions. Aristaless is a major regulator of distinct developmental processes. It is most known for its role during appendage development across animals. However, more recently other distinct biological functions have been described for this gene and its duplicates. Butterflies and moths have two copies of aristaless, aristaless1 (al1) and aristaless2 (al2), as a result of a gene duplication event. Previous work in Heliconius has shown that both copies appear to have novel functions related to wing color patterning. Here we expand our knowledge of the expression profiles associated with both ancestral and novel functions of Al1 across embryogenesis and wing pigmentation. Furthermore, we characterize Al2 expression, providing a comparative framework between gene copies within the same species, allowing us to understand the origin of new functions following gene duplication. RESULTS: Our work shows that the expression of both Al1 and Al2 is associated with the ancestral function of sensory appendage (leg, mouth, spines, and eyes) development in embryos. Interestingly, Al1 exhibits higher expression earlier in embryogenesis while the highest levels of Al2 expression are shifted to later stages of embryonic development. Furthermore, Al1 localization appears extranuclear while Al2 co-localizes tightly with nuclei earlier, and then also expands outside the nucleus later in development. Cellular expression of Al1 and Al2 in pupal wings is broadly consistent with patterns observed during embryogenesis. We also describe, for the first time, how Al1 localization appears to correlate with zones of anterior/posterior elongation of the body during embryonic growth, showcasing a possible new function related to Aristaless' previously described role in appendage extension. CONCLUSIONS: Overall, our data suggest that while both gene copies play a role in embryogenesis and wing pigmentation, the duplicates have diverged temporally and mechanistically across those functions. Our study helps clarify principles behind sub-functionalization and gene expression evolution associated with developmental functions following gene duplication events.

aristaless1 has a dual role in appendage formation and wing color specification during butterfly development.

BACKGROUND: Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae, and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets, and pharmacological manipulation of an upstream activator. RESULTS: We find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this time point. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We propose that Al1 expression in future white scales might be related to an observed downregulation of the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3-hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling. CONCLUSIONS: We propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis.

Contemporary loss of migration in monarch butterflies.

The annual migration of the monarch butterfly Danaus plexippus is in peril. In an effort to aid population recovery, monarch enthusiasts across North America participate in a variety of conservation efforts, including captive rearing and release of monarch butterflies throughout the summer and autumn. However, the impact of captive breeding on monarchs remains an open question. Here, we show that captive breeding, both commercially and by summertime hobbyists, causes migratory behavior to be lost. Monarchs acquired commercially failed to orient south when reared outdoors in the autumn, unlike wild-caught North American monarchs, yet they did enter reproductive diapause. The commercial population was genetically highly divergent from wild-caught North American monarchs and had rounder forewings, similar to monarchs from nonmigratory populations. Furthermore, rearing wild-caught monarchs in an indoor environment mimicking natural migration-inducing conditions failed to elicit southward flight orientation. In fact, merely eclosing indoors after an otherwise complete lifecycle outdoors was enough to disrupt southern orientation. Our results provide a window into the complexity-and remarkable fragility-of migration.

Disentangling Population History and Character Evolution among Hybridizing Lineages.

Understanding the origin and maintenance of adaptive phenotypic novelty is a central goal of evolutionary biology. However, both hybridization and incomplete lineage sorting can lead to genealogical discordance between the regions of the genome underlying adaptive traits and the remainder of the genome, decoupling inferences about character evolution from population history. Here, to disentangle these effects, we investigated the evolutionary origins and maintenance of Batesian mimicry between North American admiral butterflies (Limenitis arthemis) and their chemically defended model (Battus philenor) using a combination of de novo genome sequencing, whole-genome resequencing, and statistical introgression mapping. Our results suggest that balancing selection, arising from geographic variation in the presence or absence of the unpalatable model, has maintained two deeply divergent color patterning haplotypes that have been repeatedly sieved among distinct mimetic and nonmimetic lineages of Limenitis via introgressive hybridization.

The evolution and genetics of sexually dimorphic 'dual' mimicry in the butterfly Elymnias hypermnestra.

Sexual dimorphism is a major component of morphological variation across the tree of life, but the mechanisms underlying phenotypic differences between sexes of a single species are poorly understood. We examined the population genomics and biogeography of the common palmfly Elymnias hypermnestra, a dual mimic in which female wing colour patterns are either dark brown (melanic) or bright orange, mimicking toxic Euploea and Danaus species, respectively. As males always have a melanic wing colour pattern, this makes E. hypermnestra a fascinating model organism in which populations vary in sexual dimorphism. Population structure analysis revealed that there were three genetically distinct E. hypermnestra populations, which we further validated by creating a phylogenomic species tree and inferring historical barriers to gene flow. This species tree demonstrated that multiple lineages with orange females do not form a monophyletic group, and the same is true of clades with melanic females. We identified two single nucleotide polymorphisms (SNPs) near the colour patterning gene WntA that were significantly associated with the female colour pattern polymorphism, suggesting that this gene affects sexual dimorphism. Given WntA's role in colour patterning across Nymphalidae, E. hypermnestra females demonstrate the repeatability of the evolution of sexual dimorphism.

Museum genomics reveals the Xerces blue butterfly (Glaucopsyche xerces) was a distinct species driven to extinction.

The last Xerces blue butterfly was seen in the early 1940s, and its extinction is credited to human urban development. This butterfly has become a North American icon for insect conservation, but some have questioned whether it was truly a distinct species, or simply an isolated population of another living species. To address this question, we leveraged next-generation sequencing using a 93-year-old museum specimen. We applied a genome skimming strategy that aimed for the organellar genome and high-copy fractions of the nuclear genome by a shallow sequencing approach. From these data, we were able to recover over 200 million nucleotides, which assembled into several phylogenetically informative markers and the near-complete mitochondrial genome. From our phylogenetic analyses and haplotype network analysis we conclude that the Xerces blue butterfly was a distinct species driven to extinction.

The roles of hybridization and habitat fragmentation in the evolution of Brazil's enigmatic longwing butterflies, Heliconius nattereri and H. hermathena.

BACKGROUND: Heliconius butterflies are widely distributed across the Neotropics and have evolved a stunning array of wing color patterns that mediate Müllerian mimicry and mating behavior. Their rapid radiation has been strongly influenced by hybridization, which has created new species and allowed sharing of color patterning alleles between mimetic species pairs. While these processes have frequently been observed in widespread species with contiguous distributions, many Heliconius species inhabit patchy or rare habitats that may strongly influence the origin and spread of species and color patterns. Here, we assess the effects of historical population fragmentation and unique biology on the origins, genetic health, and color pattern evolution of two rare and sparsely distributed Brazilian butterflies, Heliconius hermathena and Heliconius nattereri. RESULTS: We assembled genomes and re-sequenced whole genomes of eight H. nattereri and 71 H. hermathena individuals. These species harbor little genetic diversity, skewed site frequency spectra, and high deleterious mutation loads consistent with recent population bottlenecks. Heliconius hermathena consists of discrete, strongly isolated populations that likely arose from a single population that dispersed after the last glacial maximum. Despite having a unique color pattern combination that suggested a hybrid origin, we found no genome-wide evidence that H. hermathena is a hybrid species. However, H. hermathena mimicry evolved via introgression, from co-mimetic Heliconius erato, of a small genomic region upstream of the color patterning gene cortex. CONCLUSIONS: Heliconius hermathena and H. nattereri population fragmentation, potentially driven by historical climate change and recent deforestation, has significantly reduced the genetic health of these rare species. Our results contribute to a growing body of evidence that introgression of color patterning alleles between co-mimetic species appears to be a general feature of Heliconius evolution.

Butterfly Mimicry Polymorphisms Highlight Phylogenetic Limits of Gene Reuse in the Evolution of Diverse Adaptations.

Some genes have repeatedly been found to control diverse adaptations in a wide variety of organisms. Such gene reuse reveals not only the diversity of phenotypes these unique genes control but also the composition of developmental gene networks and the genetic routes available to and taken by organisms during adaptation. However, the causes of gene reuse remain unclear. A small number of large-effect Mendelian loci control a huge diversity of mimetic butterfly wing color patterns, but reasons for their reuse are difficult to identify because the genetic basis of mimicry has primarily been studied in two systems with correlated factors: female-limited Batesian mimicry in Papilio swallowtails (Papilionidae) and non-sex-limited Müllerian mimicry in Heliconius longwings (Nymphalidae). Here, we break the correlation between phylogenetic relationship and sex-limited mimicry by identifying loci controlling female-limited mimicry polymorphism Hypolimnas misippus (Nymphalidae) and non-sex-limited mimicry polymorphism in Papilio clytia (Papilionidae). The Papilio clytia polymorphism is controlled by the genome region containing the gene cortex, the classic P supergene in Heliconius numata, and loci controlling color pattern variation across Lepidoptera. In contrast, female-limited mimicry polymorphism in Hypolimnas misippus is associated with a locus not previously implicated in color patterning. Thus, although many species repeatedly converged on cortex and its neighboring genes over 120 My of evolution of diverse color patterns, female-limited mimicry polymorphisms each evolved using a different gene. Our results support conclusions that gene reuse occurs mainly within ∼10 My and highlight the puzzling diversity of genes controlling seemingly complex female-limited mimicry polymorphisms.

Migration behaviour of commercial monarchs reared outdoors and wild-derived monarchs reared indoors.

Captive rearing of monarch butterflies is a commercial and personal pursuit enjoyed by many different groups and individuals. However, the practice remains controversial, especially after new evidence showed that both a group of commercially derived monarchs reared outdoors and a group of wild-derived but indoor-reared monarchs failed to orient south, unlike wild-derived monarchs reared outdoors. To more fully characterize the mechanisms responsible for the loss of orientation in both commercial and indoor-reared monarchs, we performed flight simulator experiments to determine (i) whether any fraction of commercial monarchs maintains a southern heading over multiple tests, and (ii) whether indoor conditions with the addition of sunlight can induce southern flight in wild-derived monarchs. Commercial monarchs changed their flight direction more often over the course of multiple tests than wild-derived monarchs. While as a group the commercial monarchs did not fly south on average, a subset of individuals did orient south over multiple tests, potentially explaining the discordance between flight simulator assays and the recovery of tagged commercial monarchs at overwintering locations. We also show that even when raised indoors with sunlight, wild-derived monarchs did not consistently orient south in the flight simulator, though wild-derived monarchs reared outdoors did orient south.

Divergence, gene flow, and the origin of leapfrog geographic distributions: The history of colour pattern variation in Phyllobates poison-dart frogs.

The geographic distribution of phenotypic variation among closely related populations is a valuable source of information about the evolutionary processes that generate and maintain biodiversity. Leapfrog distributions, in which phenotypically similar populations are disjunctly distributed and separated by one or more phenotypically distinct populations, represent geographic replicates for the existence of a phenotype, and are therefore especially informative. These geographic patterns have mostly been studied from phylogenetic perspectives to understand how common ancestry and divergent evolution drive their formation. Other processes, such as gene flow between populations, have not received as much attention. Here, we investigate the roles of divergence and gene flow between populations in the origin and maintenance of a leapfrog distribution in Phyllobates poison frogs. We found evidence for high levels of gene flow between neighbouring populations but not over long distances, indicating that gene flow between populations exhibiting the central phenotype may have a homogenizing effect that maintains their similarity, and that introgression between 'leapfroging' taxa has not played a prominent role as a driver of phenotypic diversity in Phyllobates. Although phylogenetic analyses suggest that the leapfrog distribution was formed through independent evolution of the peripheral (i.e. leapfrogging) populations, the elevated levels of gene flow between geographically close populations poise alternative scenarios, such as the history of phenotypic change becoming decoupled from genome-averaged patterns of divergence, which we cannot rule out. These results highlight the importance of incorporating gene flow between populations into the study of geographic variation in phenotypes, both as a driver of phenotypic diversity and as a confounding factor of phylogeographic inferences.

Genomic evidence for gene flow between monarchs with divergent migratory phenotypes and flight performance.

Monarch butterflies are known for their spectacular annual migration in eastern North America, with millions of monarchs flying up to 4,500 km to overwintering sites in central Mexico. Monarchs also live west of the Rocky Mountains, where they travel shorter distances to overwinter along the Pacific Coast. It is often assumed that eastern and western monarchs form distinct evolutionary units, but genomic studies to support this notion are lacking. We used a tethered flight mill to show that migratory eastern monarchs have greater flight performance than western monarchs, consistent with their greater migratory distances. However, analysing more than 20 million SNPs in 43 monarch genomes, we found no evidence for genomic differentiation between eastern and western monarchs. Genomic analysis also showed identical and low levels of genetic diversity, and demographic analyses indicated similar effective population sizes and ongoing gene flow between eastern and western monarchs. Gene expression analysis of a subset of candidate genes during active flight revealed differential gene expression related to nonmuscular motor activity. Our results demonstrate that eastern and western monarchs maintain migratory differences despite ongoing gene flow, and suggest that migratory differences between eastern and western monarchs are not driven by select major-effects alleles. Instead, variation in migratory distance and destination may be driven by environmentally induced differential gene expression or by many alleles of small effect.

The genetics of monarch butterfly migration and warning colouration.

The monarch butterfly, Danaus plexippus, is famous for its spectacular annual migration across North America, recent worldwide dispersal, and orange warning colouration. Despite decades of study and broad public interest, we know little about the genetic basis of these hallmark traits. Here we uncover the history of the monarch's evolutionary origin and global dispersal, characterize the genes and pathways associated with migratory behaviour, and identify the discrete genetic basis of warning colouration by sequencing 101 Danaus genomes from around the globe. The results rewrite our understanding of this classic system, showing that D. plexippus was ancestrally migratory and dispersed out of North America to occupy its broad distribution. We find the strongest signatures of selection associated with migration centre on flight muscle function, resulting in greater flight efficiency among migratory monarchs, and that variation in monarch warning colouration is controlled by a single myosin gene not previously implicated in insect pigmentation.

Macroevolutionary shifts of WntA function potentiate butterfly wing-pattern diversity.

Butterfly wing patterns provide a rich comparative framework to study how morphological complexity develops and evolves. Here we used CRISPR/Cas9 somatic mutagenesis to test a patterning role for WntA, a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that WntA loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, WntA is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to Vanessa forewings. In two Heliconius species, WntA specifies the boundaries between melanic fields and the light-color patterns that they contour. In the passionvine butterfly Agraulis, WntA removal shows opposite effects on adjacent pattern elements, revealing a dual role across the wing field. Finally, WntA acquired a divergent role in the patterning of interveinous patterns in the monarch, a basal nymphalid butterfly that lacks stripe-like symmetry systems. These results identify WntA as an instructive signal for the prepatterning of a biological system of exuberant diversity and illustrate how shifts in the deployment and effects of a single developmental gene underlie morphological change.

Genomic Takeover by Transposable Elements in the Strawberry Poison Frog.

We sequenced the genome of the strawberry poison frog, Oophaga pumilio, at a depth of 127.5× using variable insert size libraries. The total genome size is estimated to be 6.76 Gb, of which 4.76 Gb are from high copy number repetitive elements with low differentiation across copies. These repeats encompass DNA transposons, RNA transposons, and LTR retrotransposons, including at least 0.4 and 1.0 Gb of Mariner/Tc1 and Gypsy elements, respectively. Expression data indicate high levels of gypsy and Mariner/Tc1 expression in ova of O. pumilio compared with Xenopus laevis. We further observe phylogenetic evidence for horizontal transfer (HT) of Mariner elements, possibly between fish and frogs. The elements affected by HT are present in high copy number and are highly expressed, suggesting ongoing proliferation after HT. Our results suggest that the large amphibian genome sizes, at least partially, can be explained by a process of repeated invasion of new transposable elements that are not yet suppressed in the germline. We also find changes in the spliceosome that we hypothesize are related to permissiveness of O. pumilio to increases in intron length due to transposon proliferation. Finally, we identify the complement of ion channels in the first genomic sequenced poison frog and discuss its relation to the evolution of autoresistance to toxins sequestered in the skin.

Monarch butterflies use an environmentally sensitive, internal timer to control overwintering dynamics.

The monarch butterfly (Danaus plexippus) complements its iconic migration with diapause, a hormonally controlled developmental programme that contributes to winter survival at overwintering sites. Although timing is a critical adaptive feature of diapause, how environmental cues are integrated with genetically-determined physiological mechanisms to time diapause development, particularly termination, is not well understood. In a design that subjected western North American monarchs to different environmental chamber conditions over time, we modularized constituent components of an environmentally-controlled, internal diapause termination timer. Using comparative transcriptomics, we identified molecular controllers of these specific diapause termination components. Calcium signalling mediated environmental sensitivity of the diapause timer, and we speculate that it is a key integrator of environmental condition (cold temperature) with downstream hormonal control of diapause. Juvenile hormone (JH) signalling changed spontaneously in diapause-inducing conditions, capacitating response to future environmental condition. Although JH is a major target of the internal timer, it is not itself the timer. Epigenetic mechanisms are implicated to be the proximate timing mechanism. Ecdysteroid, JH, and insulin/insulin-like peptide signalling are major targets of the diapause programme used to control response to permissive environmental conditions. Understanding the environmental and physiological mechanisms of diapause termination sheds light on fundamental properties of biological timing, and also helps inform expectations for how monarch populations may respond to future climate change.

Frequency dependence shapes the adaptive landscape of imperfect Batesian mimicry.

Despite more than a century of biological research on the evolution and maintenance of mimetic signals, the relative frequencies of models and mimics necessary to establish and maintain Batesian mimicry in natural populations remain understudied. Here we investigate the frequency-dependent dynamics of imperfect Batesian mimicry, using predation experiments involving artificial butterfly models. We use two geographically distinct populations of Adelpha butterflies that vary in their relative frequencies of a putatively defended model (Adelpha iphiclus) and Batesian mimic (Adelpha serpa). We found that in Costa Rica, where both species share similar abundances, Batesian mimicry breaks down, and predators more readily attack artificial butterfly models of the presumed mimic, A. serpa By contrast, in Ecuador, where A. iphiclus (model) is significantly more abundant than A. serpa (mimic), both species are equally protected from predation. Our results provide compelling experimental evidence that imperfect Batesian mimicry is frequency-dependent on the relative abundance of models and mimics in natural populations, and contribute to the growing body of evidence that complex dynamics, such as seasonality or the availability of alternative prey, influence the evolution of mimetic traits.

Divergence and gene flow among Darwin's finches: A genome-wide view of adaptive radiation driven by interspecies allele sharing.

A recent analysis of the genomes of Darwin's finches revealed extensive interspecies allele sharing throughout the history of the radiation and identified a key locus responsible for morphological evolution in this group. The radiation of Darwin's finches on the Galápagos archipelago has long been regarded as an iconic study system for field ecology and evolutionary biology. Coupled with an extensive history of field work, these latest findings affirm the increasing acceptance of introgressive hybridization, or gene flow between species, as a significant contributor to adaptive evolution. Here, we review and discuss these findings in relation to both classical work on Darwin's finches and contemporary work showing similar evolutionary signatures in other biological systems. The continued unification of genomic data with field biology promises to further elucidate the molecular basis of adaptation in Darwin's finches and well beyond.

Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand.

Although animals display a rich variety of shapes and patterns, the genetic changes that explain how complex forms arise are still unclear. Here we take advantage of the extensive diversity of Heliconius butterflies to identify a gene that causes adaptive variation of black wing patterns within and between species. Linkage mapping in two species groups, gene-expression analysis in seven species, and pharmacological treatments all indicate that cis-regulatory evolution of the WntA ligand underpins discrete changes in color pattern features across the Heliconius genus. These results illustrate how the direct modulation of morphogen sources can generate a wide array of unique morphologies, thus providing a link between natural genetic variation, pattern formation, and adaptation.