Butterflies That Look Like Leaves – Nature’s Incredible Camouflage

Special resemblance of animals to natural objects such as leaves provides a representative example of evolutionary adaptation. The existence of such sophisticated features challenges our understanding of how complex adaptive phenotypes evolved. Leaf mimicry typically consists of several pattern elements, the spatial arrangement of which generates the leaf venation-like appearance. However, the process by which leaf patterns evolved remains unclear.

In this study we show the evolutionary origin and process for the leaf pattern in Kallima (Nymphalidae) butterflies. Using comparative morphological analyses, we reveal that the wing patterns of Kallima and 45 closely related species share the same ground plan, suggesting that the pattern elements of leaf mimicry have been inherited across species with lineage-specific changes of their character states. On the basis of these analyses, phylogenetic comparative methods estimated past states of the pattern elements and enabled reconstruction of the wing patterns of the most recent common ancestor. This analysis shows that the leaf pattern has evolved through several intermediate patterns. Further, we use Bayesian statistical methods to estimate the temporal order of character-state changes in the pattern elements by which leaf mimesis evolved, and show that the pattern elements changed their spatial arrangement (e.g., from a curved line to a straight line) in a stepwise manner and finally establish a close resemblance to a leaf venation-like appearance.

Our study provides the first evidence for stepwise and contingent evolution of leaf mimicry. Leaf mimicry patterns evolved in a gradual, rather than a sudden, manner from a non-mimetic ancestor. Through a lineage of Kallima butterflies, the leaf patterns evolutionarily originated through temporal accumulation of orchestrated changes in multiple pattern elements.

Evolution of complex adaptive features is a fundamental subject in evolutionary biology [1]-[4]. Central questions in relation to this subject include whether the origin of complex features was gradual or sudden, and how the evolutionary changes that generated these features accumulated over long time periods [5]-[9]. Leaf mimicry in butterfly wings (e.g. genus Kallima) provides a striking example of complex adaptive features and has led to speculation about how wing patterns evolve a close resemblance to leaves from an ancestral form that did not resemble leaves [10]-[13]. Conflicting perspectives on the evolution of leaf mimicry have led to controversial and contrasting hypotheses [14]-[19]. The origin of leaf mimicry and the process by which it evolved have not been resolved.

The genus Kallima comprises leaf butterflies that display transverse, leaf-like venation across the ventral sides of the fore- and hindwing (Figure 1a, c, d, and Figure 2 mm). The leaf pattern consists of a main vein and right- and left-sided lateral veins, each of which contain pigment elements whose spatial arrangement generates the leaf-like appearance (i.e. pigments, rather than wing veins, form the leaf-like pattern). Leaf mimicry in Kallima spp. (Kallima inachus and Kallima paralekta) was described by Wallace as ‘the most wonderful and undoubted case of protective resemblance in a butterfly’ [14]. Following this description, Darwin, Poulton, and modern evolutionary biologists have argued that the leaf mimicry pattern is a product of gradual evolution by natural selection [10],[15]-[17]. In contrast, Mivart pointed out that although leaf mimicry is assumed to be an evolutionary adaptation, its chance of establishing in a population is predicted to be low because poor mimicry of a target during the incipient stages of evolution would lead to an increased probability of predation [18]. Goldschmidt advocated the sudden emergence of leaf mimicry patterns (i.e. saltation) without intermediate forms [19]. Despite enthusiastic debate, there is as yet no direct experimental evidence for the gradual evolution of the leaf pattern.

Nymphalid ground plan and Kallima inachus leaf wing pattern. (a) When resting, K. inachus folds its fore- and hind wings and displays a leaf-like pattern to potential predators. (b) Nymphalid ground plan: This scheme consists of 11 elements. The three pairs of symmetry pattern elements include the proximal (p) and distal (d) bands designated as basal (B, blue), central (C, red), and border (BO, green) elements. Four additional elements are designated as root (R, light blue), submarginal and marginal (M, orange), discal spots (DS, yellow) and a serial array of eye spots (ESs, concentric rings). (c) Male ventral wings, resembling transverse leaf venation across fore and hind wings. (d) The leaf venation pattern is composed of several pattern elements representing a main vein and right and left lateral veins (highlighted with yellow, orange, and pink lines, respectively). (e) The Nymphalid ground plan of the K. inachus leaf pattern analysed in this study.

Nymphalid ground plan of Nymphalinae butterfly wing patterns. Using a comparative morphological approach, we dissected the extraordinarily diversified wing patterns into an assembly of Nymphalid ground plan (NGP) elements. The right, ventral wings are shown (left, each) with drawings of the NGP (right, each; mirror-opposite s of right wings). (a) Araschnia levana, (b) Mynes geoffroyi, (c) Symbrenthia hypselis, (d) Symbrenthia lilaea, (e) Hypanartia lethe, (f) Hypanartia dione, (g) Hypanartia kefersteini, (h) Vanessa cardui, (i) Vanessa atalanta, (j) Vanessa indica, (k) Antanartia delius, (l) Aglais io, (m) Aglais urticae, (n) Kaniska canace, (o) Nymphalis vau-album, (p) Polygonia c-album, (q) Polygonia c-aureum, (r) Hypolimnas bolina, (s) Precis andremiaja, (t) Precis archesia, (u) Precis octavia, (v) Junonia westermanni, (w) Junonia hierta, (x) Junonia orithya, (y) Junonia coenia, (z) Junonia lemonias, (aa) Junonia almana, (bb) Junonia atlites, (cc) Junonia iphita, (dd) Junonia erigone, (ee) Junonia hedonia, (ff) Salamis anteva, (gg) Salamis cacta, (hh) Protogoniomorpha anacardii, (ii) Protogoniomorpha parhassus, (jj) Yoma algina, (kk) Yoma sabina, (ll) Doleschallia bisaltide, (mm) Kallima paralekta. Colours are the same as those used in Figure 1.

We focused on the phylogenetic evolution of leaf mimicry patterns, for which a key principle is the ‘body plan’ or ‘ground plan’, referring to the structural composition of organisms by homologous elements shared across species [20]. Notably, butterfly wing patterns are thought to be based on a highly conserved ground plan (the Nymphalid ground plan, NGP; Figure 1b) [21]-[23]. The NGP describes the extraordinary diversification of wing patterns as modifications of an assembly of discrete pattern elements shared among species, which are suggested to be homologous and inherited across species. Previous studies have suggested the existence of the NGP in numerous species [23], including the wing patterns of leaf moths [24] and Kallima inachus [22]. The NGP has also been validated by experimental molecular data [25]. If the NGP was present in both leaf mimics and non-mimetic butterflies, this would provide an opportunity to examine the evolution of leaf mimicry from non-mimetic patterns by tracing changes in the states of NGP elements through phylogeny.

The identification of homology provides a foundation for statistical testing of the likelihood of trait evolution within a phylogenetic framework. We employed Bayesian phylogenetic inference using BayesTraits [26], which provides a platform for reconstructing ancestral states of traits [27] and for analysing the dependent evolution of state transitions [28]. Furthermore, given the rates of state transitions in traits, it is possible to assess whether changes in one trait are contingent upon the background state of another. In this analysis, contingency was defined as temporal dependency in trait evolution [29]-[31] and quantified (using the Z-score) as the degree of influence of unique, chance historical events on subsequent evolution [sensu Pagel [28],[32],[33]]. Recent studies have documented well-supported molecular phylogeny of Kallima and closely related species (tribes Nymphalini, Junoniini, and Kallimini) [34]-[36], which facilitates Bayesian phylogenetic inference.

Our objectives were to generate statistical estimation of (1) ancestral wing patterns given a lineage of leaf mimicry evolution, and (2) evolutionary process of accumulation in state changes of NGP elements. Through these analyses, we examined whether leaf mimicry evolved through gradual or sudden changes and whether these changes accumulated independently or contingently. Here, we show the evolutionary origin and process of the Kallima leaf pattern. We demonstrate that the leaf pattern is composed of an array of discrete elements described by the NGP that are also present in the wing patterns of closely related species. These results strongly suggest that evolution of the Kallima leaf pattern can be traced by changes in the states of NGP elements. We then use Bayesian phylogenetic methods to reconstruct ancestral wing patterns, and describe the evolution of leaf patterns through stepwise changes in intermediate states from the non-mimetic ancestral pattern.

The species used in this study were selected to represent major groups of Nymphalinae, which includes three higher taxa (Kallimini, Junoniini, Nymphalini). Among all genera (22 genera) comprising these three higher taxa, we selected 18 genera (Additional file 1: Figure S1). Among all species (196 species) comprising these 18 genera, we sampled 47 species (24%) (Additional file 1: Table S1). In the analyses, one major group of Nymphalinae, Melitaeini, was excluded because of very autapomorphic wing patterns [36]-[38], except for the following 4 species from 4 genera: Euphydryas phaeton, Melitaea cinxia, Phyciodes cocyta, and Chlosyne janais. Phylogenetic comparative methods assume that extant species are either completely or proportionally sampled from the taxon of interest. We thus intended to minimize the effects of biased sampling on our statistical inferences by selecting representative species sampled from almost all genera. To evaluate whether the species we selected are representative of their genus with regard to wing patterns, we checked photos of butterfly wing patterns from validated and private web sites (Additional file 2: Table S2). Because our analyses focus on geometrical characteristics (e.g., a straight line and parallel arrangement between lines) of pigmental elements forming wing patterns (Figure 3a), it is necessary to select species displaying representative wing patterns in the genus that the species belong to. Therefore, we observed the specimens and photos to determine whether the 11 characteristic states of the NGP used for phylogenetic comparative analyses are typical of the genera. We checked 116 species (89% of all 131 species) and confirmed the unbiased selection of the species used in this study. For example, in the genus Kallima, the two species we selected (Kallima inachus and paralekta) appeared to be representative to this genus because they exhibited wing patterns similar with to those of another species (Kallima alompra) with regard to the Nymphalid ground plan (NGP; see Figures 1b, 3a) (Additional file 1: Figure S2). Thus, although the results should be interpreted cautiously, we are confident that by applying unbiased sampling of species from most genera, we conducted a practical estimation of the evolution of wing patterns.

Bayesian inference of ancestral character – state reconstruction of wing pattern evolution. (a) Leaf vein features of the Kallima wing pattern were coded as 11 characters (Ch) as follows: Ch 1: parallelism of DS and B; Ch 2: attachment of DS and Cp; Ch 3: Cd a single broken straight line; Ch 4: bending of BOp to distal side; Ch 5: straightness of upper side of BOp; Ch 6: vestigiality of ESs; Ch 7: vestigiality of B; Ch 8: fragmentation of Cp; Ch 9: vestigiality of DS; Ch 10: straightness of Cd; Ch 11: vestigiality of ESs. These characters were also surveyed in the closely related species and coded as one of two binary states (‘state 1’ = Kallima-like state; ‘state 0’ = non-Kallima-like state). Characters in forewings (squares) and hindwings (circles) are coloured as in Figure 1. (b) Reconstructed ancestral character states are represented as pie charts indicating Bayesian posterior probability at four selected nodes (A, B, C, and D) by shaded circles (black = state 1; grey = state 0). In the molecular phylogeny, genus Kallima is highlighted in the red box. B, Cp, Cd, BOp, DS, and ESs are defined and presented in Figure 1.

To take phylogenetic and branch-length uncertainty into account in our analyses, we generated Bayesian trees by combining three recently published datasets [34],[36],[39] and confirmed that our phylogeny was consistent with that proposed previously (Additional file 1: Figure S3). We used eight nuclear (wingless, ef-1α, RpS5, GADPH, ArgKin, CAD, IDH and MDH) and one mitochondrial (cox1) gene sequences to reconstitute the phylogenetic tree of the species included in the analysis. Multiple alignment was performed using ClustalW [40] in MEGA5 [41] as previously described [42]. In brief, we aligned the nucleotide sequences based on their translated amino acid sequences, and the aligned sets of genes were concatenated for use in subsequent analyses. Species names and GenBank accession numbers of sequences used in this study are provided in Additional file 1: Table S3. The original s of voucher specimens are cited in the NSG’s DNA sequences database (http://nymphalidae.utu.fi/db.php). Six species (Adelpha bredowii, Apatura iris, Asterocampa idyja, Eurytela dryope, Hamadryas februa, and Heliconius hecale) were used as the outgroup taxa. We constructed datasets composed of 7,342 nucleotide sites from nine concatenated genes.

We used PartitionFinder [43] to identify nucleotide substitution models and partitioning strategies for the dataset. Breaking down the nucleotide data by codon position resulted in 27 partitions (the first, second, and third codon positions for each gene), which were combined to result in nine partitions. A nucleotide substitution model was selected for each partition using the number of sites as the sample size based on the Bayesian information criterion (BIC) (see Additional file 3: Data S1: the attached nexus file for the alignment, partitioning and substitution models). The sequence data as well as phylogenetic analysis are also available at TreeBASE (Submission ID: 16541). We used MrBayes 3.1.2 for the Bayesian inference of phylogenetic trees, which includes the assumption of proportional branch length among the partitions. We ran four concurrent analyses of 2 × 107 generations with eight chains each (seven heated and one cold) using different random starting trees, and sampled every 100 generations. Runs of all procedures were checked for stationarity, convergence, and adequate mixing of the Markov chains using Tracer version 1.5 [44]. From each data set, we discarded the first 60,000 samplings as burn-in and combined the resulting MCMC tree samples for subsequent estimation of posteriors.

Bayesian phylogenetic inference of ancestral wing patterns

The results of our comparative morphological analyses explain the evolution of the Kallima leaf patterns, which are formed of NGP elements with specific modifications that confer a leaf-like appearance. A Bayesian phylogenetic method was used to reconstruct the ancestral states of the butterfly wing patterns at phylogenetic nodes (A–D in Figure 3b). We coded the K. inachus wing pattern using 11 prominent characters from the suite of characteristics that formed the leaf-like appearance (Figure 3a). This coding was also performed on the closely related species and their wing patterns were characterized as one of two binary states (Additional file 1: Tables S4 and S5). Analyses implemented in BayesTraits account for uncertainty in phylogeny and branch length; we reconstructed phylogenetic trees by combining three previously published datasets [34],[36],[39] (Figure 3b; Additional file 1: Figure S3) and obtained results that were consistent with previous reports [34],[36],[39]. The 11 character states at node A were estimated as follows in the forewing: the Cp and DS elements were not attached (Ch 2), the Cd element did not form a single, broken straight line (Ch 3), the BOp was ordinarily curved (Ch 4 and 5), and the ESs were not vestigial (Ch 6). In the hind wing, the B, DS, and ESs were not vestigial (Ch 7, 9, and 11), the Cp element was not fragmented (Ch 8), and the Cd element did not form a straight line (Ch 10) (Figure 3b). Taken together, these results strongly suggested that the most ancestral pattern was a non Kallima-like pattern.

Our analyses revealed further evolution of wing patterns (Figure 3b). At node B of the phylogeny, the character states were reconstructed such that DS in the hindwing became vestigial (Ch 9) and the Cd in the hindwing became straightened (Ch 10). The overall wing pattern evolved through the accumulation of changes from the most ancestral wing pattern at node A. Then, at node C, the Cd in the forewing changed to a single broken line (Ch 3), the B in the hindwing was vestigial (Ch 7), and the Cp element in the hindwing became fragmented (Ch 8). The wing pattern evolved through additional changes that caused some characteristics to transition to a Kallima-like state. Finally, at node D, all character states had transitioned to the Kallima-like state (state ‘1’). These analyses demonstrate that the overall leaf pattern originated via stepwise transitions through intermediate forms. These results clearly showed that, at the very least, this evolutionary transition did not occur suddenly.

Estimation of common ancestral states at phylogenetic nodes

Reconstruction of ancestral character states was performed in a Bayesian framework using BayesTraits ver. 2.0 (www.evolution.rdg.ac.uk/BayesTraits.html) [26]. In contrast to the optimality criterion (parsimony and likelihood), the Bayesian Markov chain Monte Carlo (MCMC) method has the advantage of investigating the uncertainty of the phylogeny and the parameters of the model for trait evolution [27]. BayesTraits implements the program MULTISTATE, which calculates the posterior probability of states in all nodes across the posterior distribution of trees that are hypothetical ancestors of the taxa of interest. This calculation uses reversible-jump (rj)-MCMC simulations to combine uncertainty about the existence of a node and its character state, which enables sampling of all possible models of evolution (rather than just the rate parameters as in conventional MCMC) in proportion to their posterior probabilities [28],[51]. Reconstructions were performed using the most recent common ancestor (MRCA) approach; when the node of interest did not exist, the minimal node that contained all terminal taxa of the clade defined by our node of interest (plus one or more extra taxa) was reconstructed instead. In these analyses, polymorphic character states were accounted for, as they were considered as occurrences with an equivalent probability for calculation [26].

To run the rj-MCMC chain, 4,000 trees were subsampled from each of the four codon-partitioned MrBayes runs (a total of 2 × 105 trees). To allow adequate mixing and achievement of stationary, the rj-MCMC chain was run for 5.005 × 107 iterations with the first 5 × 104 iterations discarded as burn-in and a sampling interval of 1000 iterations, for a final sample of 5 × 104 iterations. We used a uniform prior for the analyses. To avoid autocorrelation and to allow exploration of ample parameter space, the ratedev parameter was automatically adjusted for each analysis to maintain an acceptance rate of 30%, to vary the amount by which the rate parameters were allowed to change between iterations of the Markov chain (ratedev), as recommended in the BayesTraits manual [26]. We examined the output in Tracer version 1.5 [44] to confirm the stationarity of the log-likelihood. Manipulation of trees was conducted using the ‘ape’ package [52] in R.

How Caterpillars Turn Into Butterflies

FAQ

What type of butterfly looks like a leaf?

The question of how the closed wings of dead leaf (or oakleaf) butterflies from the Kallima genus came to perfectly resemble brown leaves—from their veins down to tiny fungus spots—has been hotly debated. (See “Photos: Masters of Disguise-Amazing Insect Camouflage.”)

What are the leaves like butterflies?

Oakleaf butterflies of the genus Kallima occur in mountains and lowland regions across Asia. The wing patterns of these butterflies vary across species but they all look like dead, brown leaves.

What are the butterflies that look like brown leaves?

Kallima inachus, the orange oakleaf, Indian oakleaf or dead leaf, is a nymphalid butterfly found in Tropical Asia from India to Japan. With wings closed, it closely resembles a dry leaf with dark veins and is a commonly cited example of camouflage.

Where do orange oakleaf butterflies live?

The Dead Leaf Butterfly, aka Kallima inachus or the orange oakleaf butterfly, lives in Tropical Asia, like India, Thailand, Vietnam, and Japan. They feed on sap, over-ripe fruit, and nutrients found in puddles (this is called mud-puddling).

What plants look like butterflies?

So, here is a brief rundown of plants that look like butterflies, including their most prominent features: Red butterfly wing plant – has leaves that flutter like butterflies in the wind. Blue butterfly bush – a tropical shrub that can grow up to 10 feet high with blue butterfly-shaped flowers.

Do Butterflies look like leaves?

Butterflies are known for their colored wings. These wings can sometimes make them look like leaves. Some of them are so similar to leaves that they’re even confused with a leaf. Oakleaf butterflies are commonly confused with the leaves of oak trees. Here are a few common butterflies that look like leaves. 1. Dry-leaf Commodore

What do autumn leaf butterflies look like?

The colors of the autumn leaf butterfly vary, but they typically include a combination of brown, orange, and black, closely mirroring the hues seen on fallen leaves. The autumn leaf butterfly exhibits behaviors that enhance its leaf mimicry. When resting, the butterfly folds its wings up, exposing the undersides.

What do green butterflies look like?

Green butterflies are some of the most common colorful butterflies around the world. They often use their green color for camouflage, with many species having green ventral coloring on their wings that makes them look like plant or tree leaves.

What wing plant resembles a butterfly?

1. Red Butterfly Wing Plant (Christia Vespertilionis) The red butterfly wing plant or mariposa leaves closely resemble Troides brookiana, or “birdwing butterflies,” in mid-flight due to their paired elongated, horizontal foliage and rounded points. The plant’s leaves also resemble flying butterflies when they are moved by the wind.

What insects look like leaves?

As you can see, there are many insects in the world that look like leaves–and this is only a partial list. Some of the insects on this list include various butterflies, moths, praying mantises, katydids, and leaf insects.