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.

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.

Rapid diversification associated with ecological specialization in Neotropical Adelpha butterflies.

Rapid diversification is often associated with morphological or ecological adaptations that allow organisms to radiate into novel niches. Neotropical Adelpha butterflies, which comprise over 200 species and subspecies, are characterized by extraordinary breadth in host plant use and wing colour patterns compared to their closest relatives. To examine the relationship between phenotypic and species diversification, we reconstructed the phylogenetic history of Adelpha and its temperate sister genus Limenitis using genomewide restriction-site-associated DNA (RAD) sequencing. Despite a declining fraction of shared markers with increasing evolutionary distance, the RAD-Seq data consistently generated well-supported trees using a variety of phylogenetic methods. These well-resolved phylogenies allow the identification of an ecologically important relationship with a toxic host plant family, as well as the confirmation of widespread, convergent wing pattern mimicry throughout the genus. Taken together, our results support the hypothesis that evolutionary innovations in both larvae and adults have permitted the colonization of novel host plants and fuelled adaptive diversification within this large butterfly radiation.

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.

Mutualistic mimicry and filtering by altitude shape the structure of Andean butterfly communities.

Both the abiotic environment and abiotic interactions among species contribute to shaping species assemblages. While the roles of habitat filtering and competitive interactions are clearly established, less is known about how positive interactions, whereby species benefit from the presence of one another, affect community structure. Here we assess the importance of positive interactions by studying Andean communities of butterflies that interact mutualistically via Müllerian mimicry. We show that communities at similar altitudes have a similar phylogenetic composition, confirming that filtering by altitude is an important process. We also provide evidence that species that interact mutualistically (i.e., species that share the same mimicry wing pattern) coexist at large scales more often than expected by chance. Furthermore, we detect an association between mimicry structure and altitude that is stronger than expected even when phylogeny is corrected for, indicating adaptive convergence for wing pattern and/or altitudinal range driven by mutualistic interactions. Positive interactions extend far beyond Müllerian mimicry, with many examples in plants and animals, and their role in the evolution and assembly of communities may be more pervasive than is currently appreciated. Our findings have strong implications for the evolution and resilience of community structure in a changing world.

Convergent flight morphology among Müllerian mimic mutualists.

Müllerian mimicry involves a signal mutualism between prey species, shaped by visually hunting predators, and recent work has emphasized the importance of color pattern. Predators respond to more than color pattern, however, and other traits are much less studied. This article examines the hypothesis of convergent evolution in flight-related morphology among eight mimicry complexes composed of 51 butterfly species (Nymphalidae, Danainae, Ithomiini) from a single community in Ecuador. Phylogenetic comparative analyses of 14 variables indicated strong morphological differences between mimicry complexes belonging to three clusters of morphological space ("large yellow transparent," "tiger," and "transparent"), not the eight predicted based on color pattern alone. Analyses found convergence within mimicry complexes, convergence between mimicry complexes within morphospace clusters, and divergence between mimicry complexes from different morphospace clusters. These three clusters differed in size, and body and wing shape, predicting that flight biomechanics also converge (i.e., locomotor mimicry). Potential constraints on evolution of morphological mimicry related to predator discrimination, and evolutionary rates, likely e xplain why flight-related morphology differences were limited to three clusters of morphological space. Finally, the added complexity that flight-related morphology brings to signals between predator and prey indicates that evolutionary switches in color pattern are not all equally likely, potentially limiting the evolution of color patterns if they do not match morphology.

Vertical distribution, flight behaviour and evolution of wing morphology in Morpho butterflies.

1. Flight is a key innovation in the evolution of insects that is crucial to their dispersal, migration, territoriality, courtship and predator avoidance. Male butterflies have characteristic territoriality and courtship flight behaviours, and females use a characteristic flight behaviour when searching for host plants. This implies that selection acts on wing morphology to maximize flight performance for conducting important behaviours among sexes. 2. Butterflies in the genus Morpho are obvious components of neotropical forests, and many observations indicate that they show two broad categories of flight behaviour and flight height. Although species can be categorized as using gliding or flapping flight, and flying at either canopy or understorey height, the association of flight behaviour and flight height with wing shape evolution has never been explored. 3. Two clades within Morpho differ in flight behaviour and height. Males and females of one clade inhabit the forest understorey and use flapping flight, whereas in the other clade, males use gliding flight at canopy level and females use flapping flight in both canopy and understorey. 4. We used independent contrasts to answer whether wing shape is associated with flight behaviour and height. Given a single switch to canopy habitation and gliding flight, we compared contrasts for the node at which the switch to canopy flight occurred with the distribution of values in the two focal clades. We found significant changes in wing shape at the transition to canopy flight only in males, and no change in size for either sex. A second node within the canopy clade suggests that other factors may also be involved in wing shape evolution. Our results reinforce the hypothesis that natural selection acts differently on male and female butterfly wing shape and indicate that the transition to canopy flight cannot explain all wing shape diversity in Morpho. 5. This study provides a starting point for characterizing evolution of wing morphology in forest butterflies in the contexts of habitat selection and flight behaviour. Further, these observations suggest that exploring wing shape evolution for canopy and understorey species in other insects may help understand the effects of habitat destruction on biological diversity.

The immature stages and natural history of Veladyris pardalis (Salvin, 1869) in Eastern Ecuador (Lepidoptera: Nymphalidae: Lthomiinae).

Abstract We describe the immature stages and oviposition behavior of Veladyris pardalis (Salvin, 1869) from northeastern Ecuador. An unidentified species of Solanum (Solanaceae) is the larval food plant. Eggs are laid singly on leaves, stems or epiphytes growing on the host. Veladyris pardalis has four larval stadia, and takes 64-70 days to mature from oviposition to adult.

Phylogenomic Analyses Clarify True Species within the Butterfly Genus Speyeria despite Evidence of a Recent Adaptive Radiation.

When confronted with an adaptive radiation, considerable evidence is needed to resolve the evolutionary relationships of these closely related lineages. The North American genus Speyeria is one especially challenging radiation of butterflies due to potential signs of incomplete lineage sorting, ongoing hybridization, and similar morphological characters between species. Previous studies have found species to be paraphyletic and have been unable to disentangle taxa, often due to a lack of data and/or incomplete sampling. As a result, Speyeria remains unresolved. To achieve phylogenetic resolution of the genus, we conducted phylogenomic and population genomic analyses of all currently recognized North American Speyeria species, as well as several subspecies, using restriction-site-associated DNA sequencing (RADseq). Together, these analyses confirm the 16 canonical species, and clarify many internal relationships. However, a few relationships within Speyeria were poorly supported depending on the evolutionary model applied. This lack of resolution among certain taxa corroborates Speyeria is experiencing an ongoing adaptive radiation, with incomplete lineage sorting and lack of postzygotic reproductive barriers contributing to hybridization and further ambiguity. Given that many Speyeria taxa are under duress from anthropogenic factors, their legal protection must be viewed cautiously and on a case by case basis in order to properly conserve the diversity being generated.

Larval Food Limitation in a Speyeria Butterfly (Nymphalidae): How Many Butterflies Can Be Supported?

For herbivorous insects the importance of larval food plants is obvious, yet the role of host abundance and density in conservation are relatively understudied. Populations of Speyeria butterflies across North America have declined and Speyeria adiaste is an imperiled species endemic to the southern California Coast Ranges. In this paper, we study the link between the food plant Viola purpurea quercetorum and abundance of its herbivore Speyeria adiaste clemencei to better understand the butterfly's decline and aid in restoration of this and other Speyeria species. To assess the degree to which the larval food plant limits adult abundance of S. a. clemencei in 2013, we compared adult population counts to population size predicted from a Monte Carlo simulation using data for number of V. pur. quercetorum plants, number of leaves per plant, and leaf area per plant, with lab estimates of leaf area consumed to reach pupal stage on the non-native host V. papilionacea. Results indicated an average estimate of 765 pupae (median = 478), with 77% of the distribution being <1000 pupae. However, this was heavily dependent on plant distribution, and accounting for the number of transect segments with sufficient host to support a pupa predicted 371 pupae. The adult population empirical estimate was 227 individuals (95% CI is 146 to 392), which lies near the first quartile of the simulated distribution. These results indicate that the amount of host available to larvae was more closely linked to adult abundance than the amount of host present, especially when considering assumptions of the analyses. The data also indicate that robust populations require host density well in excess of what is eaten by larvae, in combination with appropriate spacing, to mitigate factors such as competition, starvation from leaving host patches, or unrelated to food plant, such as mortality from drought, predators, parasites, or disease.

Testing the adaptive hypothesis of Batesian mimicry among hybridizing North American admiral butterflies.

Batesian mimicry is characterized by phenotypic convergence between an unpalatable model and a palatable mimic. However, because convergent evolution may arise via alternative evolutionary mechanisms, putative examples of Batesian mimicry must be rigorously tested. Here, we used artificial butterfly facsimiles (N = 4000) to test the prediction that (1) palatable Limenitis lorquini butterflies should experience reduced predation when in sympatry with their putative model, Adelpha californica, (2) protection from predation on L. lorquini should erode outside of the geographical range of the model, and (3) mimetic color pattern traits are more variable in allopatry, consistent with relaxed selection for mimicry. We find support for these predictions, implying that this convergence is the result of selection for Batesian mimicry. Additionally, we conducted mark-recapture studies to examine the effect of mimicry and found that mimics survive significantly longer at sites where the model is abundant. Finally, in contrast to theoretical predictions, we found evidence that the Batesian model (A. californica) is protected from predation outside of its geographic range. We discuss these results considering the ongoing hybridization between L. lorquini and its sister species, L. weidemeyerii, and growing evidence that selection for mimicry predictably leads to a reduction in gene flow between nascent species.

Aristaless Controls Butterfly Wing Color Variation Used in Mimicry and Mate Choice.

Neotropical Heliconius butterflies display a diversity of warningly colored wing patterns, which serve roles in both Müllerian mimicry and mate choice behavior. Wing pattern diversity in Heliconius is controlled by a small number of unlinked, Mendelian "switch" loci [1]. One of these, termed the K locus, switches between yellow and white color patterns, important mimicry signals as well as mating cues [2-4]. Furthermore, mate preference behavior is tightly linked to this locus [4]. K controls the distribution of white versus yellow scales on the wing, with a dominant white allele and a recessive yellow allele. Here, we combine fine-scale genetic mapping, genome-wide association studies, gene expression analyses, population and comparative genomics, and genome editing with CRISPR/Cas9 to characterize the molecular basis of the K locus in Heliconius and to infer its evolutionary history. We show that white versus yellow color variation in Heliconius cydno is due to alternate haplotypes at a putative cis-regulatory element (CRE) downstream of a tandem duplication of the homeodomain transcription factor aristaless. Aristaless1 (al1) and aristaless2 (al2) are differentially regulated between white and yellow wings throughout development with elevated expression of al1 in developing white wings, suggesting a role in repressing pigmentation. Consistent with this, knockout of al1 causes white wings to become yellow. The evolution of wing color in this group has been marked by retention of the ancestral yellow color in many lineages, a single origin of white coloration in H. cydno, and subsequent introgression of white color from H. cydno into H. melpomene.

Limited performance of DNA barcoding in a diverse community of tropical butterflies.

DNA 'barcoding' relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5' half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet.

Mitochondrial DNA barcoding detects some species that are real, and some that are not.

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.

Polymorphic butterfly reveals the missing link in ecological speciation.

Ecological speciation occurs when ecologically based, divergent selection causes the evolution of reproductive isolation. There are many empirical examples of this process; however, there exists a poorly characterized stage during which the traits that distinguish species ecologically and reproductively segregate in a single population. By using a combination of genetic mapping, mate-choice experiments, field observations, and population genetics, we studied a butterfly population with a mimetic wing color polymorphism and found that the butterflies exhibited partial, color-based, assortative mate preference. These traits represent the divergent, ecologically based signal and preference components of sexual isolation that usually distinguish incipient and sibling species. The association between behavior and recognition trait in a single population may enhance the probability of speciation and provides an example of the missing link between an interbreeding population and isolated species.

Temperature-dependent oxygen limitation in insect eggs.

Most terrestrial insect embryos support metabolism with oxygen from the environment by diffusion across the eggshell. Because metabolism is more temperature sensitive than diffusion, embryos should be relatively oxygen-limited at high temperatures. We tested whether survival, development time and metabolism of eggs of a moth, Manduca sexta, were sensitive to experimentally imposed variation in atmospheric oxygen availability (5-50 kPa; normoxia at sea level is 21 kPa) across a range of biologically realistic temperatures. Temperature-oxygen interactions were apparent in most experiments. Hypoxia affected survival more strongly at warmer temperatures. Metabolic rates, measured as rates of CO2 emission, were virtually insensitive to hypo- and hyperoxia at 22 degrees C but were strongly influenced at 37 degrees C. Radial profiles of P(O2) inside eggs, measured using an oxygen microelectrode, demonstrated that 3-day-old eggs had broad central volumes with P(O2) less than 2 kPa, and that higher temperature led to lower P(O2). These data indicate that at realistically high temperatures (32-37 degrees C) eggs of M. sexta were oxygen limited, even in normoxia. This result has important implications for insect population ecology and the evolution of eggshell structures, and it suggests a novel hypothesis about insect gigantism during Paleozoic hyperoxia.