Heh, I haven't even had chance to cover the really remarkable papers in the collection, such as the speciation experiment by Diane Dodd back in 1989, which can be replicated in any well equipped high school laboratory, and which establishes the validity of the basic postulates of allopatric speciation. In short, conduct this experiment properly, and in three years, you can produce a new species of fruit fly in your greenhouse. Then there's a nice paper on Heliconius butterflies in the collection, in which the authors replicated a wild speciation event in the laboratory. I'll have fun presenting that one at some point in the future. :)

By the way, I note with interest that the two genes mentioned in that paper, namely invected and distal-less, are cited in later papers (for which I gave citations in earlier posts, incidentally) as being instrumental in wing pattern control in butterflies. Ah, don't you just love scientific consilience of this sort?

Time, methinks, to take a more in depth look at the Kallima wing pattern paper.

But before I do, in the interests of combating the creationist duplicity I've observed all too frequently in my travels, it's apposite to cover some ground, with respect to the structure of a scientific paper. Authors of scientific papers follow a well-defined set or rules when compiling papers for submission, dividing the contents of the paper into specific sections, and in the case of the first section, the abstract, this frequently exhibits a particular form of exposition of the contents. That form consists of the following steps:

[1] Present a brief summary of the state of knowledge before the submission of the new research in the paper, including the outstanding question that the newly submitted research is intended to answer;

[2] Summarise the experimental findings the authors have covered in more details in the later sections of the paper, and why those findings provide an answer to the outstanding question in step [1] above;

[3] Present the conclusion derived from the findings in step [2] above (the reasons why that conclusion is a robust conclusion to draw, appearing at the end of the paper in the Discussion section).

All too often, one of the more mendacious approaches taken by creationists to the emergence of embarrassing scientific papers, is to quote mine them for comments contained therein, that are pertinent to the state of knowledge before submission of the new research, whilst ignoring completely the rest of the paper, in which that new research is expounded, and the answers to the outstanding questions presented. Another favourite creationist trick is to subject to rampant apologetic abuse, the language used in the paper, which is frequently couched using what I term "the scientific subjunctive". This form of language presents the new findings, in the honestly tentative manner that scientists always present their findings to other scientists, with frequent appearance of subjunctive forms such as "may" or "might" in the text. This language is adopted, precisely because the research is new, and is being submitted to other scientists for peer review, with the intent that once the paper is accepted for publication, that research becomes a new addition to the prior art that can be cited by future authors. In short, the language is chosen to make explicit the basic approach, "here is our new data and experimental results, here are the conclusions we draw therefrom, and the reasons we consider said conclusions to be robust", the idea being that it is left to other reviewers to decide whether they agree with this, said agreement being signalled by publication of the paper.

The moment the paper is published, however, it is then understood that appropriate, knowledgeable reviewers have agreed with the authors, that the results do indeed lead to the stated conclusions, and that the work in question is a proper addition to the grand scientific lexicon. So let's kill off right from the start, the duplicitous apologetic misuse of scientific papers by creationist pedlars of worthless apologetics, before moving on to the exposition of the actual paper.

So, time for the paper proper. The citation is as follows:

Gradual And Contingent Evolutionary Emergence Of Leaf Mimicry In Butterfly Wing Patterns by Takao R. Suzuki, Shuichiro Tomita and Hideki Sezutsu, BMC Evolutionary Biology, 14:229-241 (25th November 2014) [Full paper downloadable from here]

Let's begin with that abstract I've described above (additional emphases mine):

Abstract

Background

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.

Results

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.

Conclusions

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.

Note the format I described above - [1], Here's a currently unanswered question; [2] Here's the work we did to answer that question; [3] Here's our answer.

So, we move on to the actual meat of the paper ...

Background

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.

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.

So, immediately, after expounding a little on the history of the topic, the authors move to the matter of how to answer the question, citing prior research (the numbers in square brackets tally with a list of papers at the end) establishing that there exists what is known as the "Nymphalid Ground Plan", a collection of spatially arranged pattern elements on the wings of Lepidoptera in this Family (other ground plans for other Families also exist), and that this ground plan has been found to be present in every species thus far studied. As a consequence, the presence of this ground plan would allow for the arrangement of pattern elements between different species to be analysed, and the specific modification of that ground plan for each species mapped. Work could then, of course, be pursued detecting the genetic basis of that gound plan (citation [25] in the papers list is for a paper covering some of the work in this field), and the relevant genetic differences in each lineage could then be elucidated. This would provide a powerful insight into the manner in which these wing patterns are generated.

Furthermore, since all of these butterflies share a common ancestor, the genetic data would allow a phylogeny to be constructed, which is, in effect, an inheritance tree mapping the steps from common ancestor to present day lineages. Once that tree is constructed, scientists can then work backwards, determine the likely wing pattern possessed by the original common ancestor, and determine how each modern, extant lineage acquired its particular pattern in a stepwise fashion.

I'll pause for a moment to address the "imperfect mimic" question alluded to above, which has actually been answered quite neatly by another paper in my collection, in which it was determined through experiment that even a poor quality mimic will have sufficient additional advantage to be selectable, to the point where the new mimetic feature becomes fixed in the population. This can then, of course, be built upon, by the emergence of new features improving the degree of mimicry. This point being addressed, of course, in the interests of preventing any anticipated quote mining.

So, let's see how this was done, shall we?

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.

So, the steps consist of:

[1] Map the pattern element arrangements in each species in the phylogeny;

[2] Use these to develop a phylogenetic tree, which will have the common ancestor at its base, including the likely pattern arrangement of the common ancestor, and the patterns of intermediates leading to the modern species;

[3] Use that tree to determine the steps leading from the ancestral pattern to the modern day pattern.

Of course, the actual work involved to achieve this end is pretty intricate, but that's what scientists are paid to do.

So, we move on, and see how this was done ...

Methods

Sampling strategy

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.

So, step one, pick the species to be used in the analysis, taking care to eliminate any sampling biases. The next paragraphs cover the technical details of setting up a phylogeny, and don't need to be quoted in full in order to enhance our understanding of the process, but basically, the authors selected relevant pattern elements appearing in all species in the phylogeny, and used those as the basis for constructing the tree. It's worth covering in some detail what those characters are, and for this, we have to turn to the construction of the basic Nymphalid Ground Plan (NGP), which consists of three distinct major spatial zones (there's a nice illustration in the paper of these) - namely, the basal symmetry zone (closest to the wing root), the central symmetry zone and the border/ocelli symmetry zone. A fourth, narrower region, the marginal symmetry zone, is found near the wing edges. Within the basal symmetry zone, there are two bands, the proximal and distal bands, marking the effective boundaries of that zone, and there is also a proximal and distal band marking the boundaries of the other two symmetry zones. Each wing also has a discal cell, with associated mobile elements, known collectively as the baso-discal complex, which manifests as markings within the discal cell of the wing. The eye spots in the border/ocelli symmetry zone are nucleation sites within which genes controlling eyespot formation are expressed. Note that 'proximal' means closest to the wing root, and 'distal' means closest to the wing edge.

So referring to the diagram in Figure 1 of the paper, the character traits found in Kallima are:

[1] Parallel alignment of discal spot and border symmetry zone;

[2] Attachment of discal spot to central symmetry zone proximal band;

[3] Central symmetry zone distal band a single, unbroken straight line;

[4] Bending of border symmetry zone proximal band to distal side;

[5] Upper side of border symmetry zone proximal band straight;

[6] Eyespots in forewing border symmetry zone vestigial;

[7] Fragmentation of central symmetry zone proximal band in hindwing;

[8] Vestigial basal symmetry zone in hindwing;

[9] Vestigial discal spot in hindwing;

[10] Central symmetry zone distal band straight;

[11] Eyespots in hindwing border symmetry zone vestigial;

Now, it's worth noting at this point, that there are numerouswhich have been demonstrated via appropriate laboratory experiments to be governed by a range of genes, including invected, engrailed, distal-less, aristaless2, wingless, wntA and optix, among several others. Many of these genes are involved in spatial pattern arrangement, whilst optix is involved in colour-filling of spatial elements. I've already cited the papers containing these results in past posts, so further citation is superfluous at this juncture. Different lineages possess modifications of the genes in question, facilitating shifting of elements and changes of colour used to fill those elements. For example, wntA and wingless are expressed in the baso-discal complex and the marginal symmetry zone, with wntA further expressed in the central symmetry zone, these being determined by experimental gene knockout of the genes in question in target species, resulting in observable changes in the patterns emerging in the affected zones. Likewise, optix knockout experiments result in colour changes in the pattern elements. So, we already have a wealth of knowledge concerning the underlying molecular biology of butterfly wing patterns, and courtesy of the relevant experimental results, this is no longer in any doubt.

Moving on, a couple of details about the tree construction ...

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].

Basically, the software used to construct the tree from the data, was able to determine additional nodes in the tree where these were not directly specified by the data, by determining the probabilities of multiple possible alternatives, and selecting the most probable ones to be members of the final tree.

Moving on, a brief word about the statistical modus operandi in action, for those familiar with the topic of Bayesian analysis ...

Estimation of dependent evolution

We used the DISCRETE program implemented in BayesTraits [26],[28] to test for (in)dependence of pairs of character state changes. As described above, this method also controls for uncertainty of phylogeny and model parameters for trait evolution [28]. The BayesDiscrete model describes changes in two dichotomous traits over branches of a phylogenetic tree via a continuous-time Markov process. The parameters of the trait evolution model represent the values of the transition rates between the eight possible character states in a model of dependent evolution. Eight rate parameters constitute the dependent model, which assumes that each character evolves (shifts forwards and backwards) at different rates depending on the state of the second character. In the independent model, forward and backward shifts in one character occur at the same rate regardless of the state of the second parameter (i.e. q12 = q34, q13 = q24, q21 = q43, and q31 = q42). Hence, a model of independent evolution has four parameters. The dependence of two traits can be investigated by comparing an independent model (in which traits evolve independently) with a dependent model where traits evolve in a correlated fashion. These models can be compared from the logarithmic Bayes Factor (log-BF), calculated as [2(log[harmonic mean (dependent model)] – log[harmonic mean (independent model)]) [26]. The harmonic means of the log-likelihoods converge on the marginal likelihoods after an adequately long run of the Markov chain [53], and therefore can be used in calculating the Bayes factors. Following Pagel and Meade [26], a result of greater than 2 approximated by the harmonic means from the final iteration of the MCMC runs was considered to represent evidence favouring the dependent model (Additional file 1: Table S6).

Now we get to the meat of the paper, namely, determining the order of changes of those 11 characters!

Estimation of order of accumulation

The posterior probability distributions of the eight transition parameters in the dependent evolutionary model were used to estimate whether the change in one state was contingent upon the background state of the other state [26],[28]. This calculation was made from the proportion of evolutionary models analysed by rj-MCMC for which a value of zero was assigned to the transition parameter (Z-score) [28],[32],[33]. If the value of one transition rate parameter in the dependent model shows a higher Z-score, that transition is less likely to occur. Thus, the Z-score represents a degree of restriction, where evolutionary trajectories or pathways are constrained. Contingency of evolutionary changes was evaluated by comparing critical pairs of parameters (i.e. q12 vs. q34; q13 vs. q24). For example, if q12 (the rate parameter for the [0, 0] → [0, 1] transition) shows a higher Z-score, but q34 (the rate parameter for the [1, 0] → [1, 1] transition) shows a lower Z-score, then evolutionary change of the second character from 0 to 1 is more likely to occur when the background state of the first character =1. This evolutionary case can be interpreted to indicate that change in the second character is contingent upon change in the first character. In the Bayesian phylogenetic method, contingency in changes in character states was detected by a bias between sets of two transition rates (Additional file 1: Table S7 and Figure S4).

Estimation of contingency was conducted within pairs of character states that showed dependent evolution (Additional file 1: Table S8). Contingency between a pair of character state changes was determined from the results of the model with a gamma hyperprior and evaluated according to relatively low transition rates (Z-score >70%) [32],[54],[55]. The contingency between all pairs of character state changes was summarized in the form of a network (Figure 4c), in which nodes represent events in the changes of character states and arrowed links represent the order of accumulation of character state changes.

So, having determined the likely dependencies between the character state changes, the authors deliver their admittedly long coup de grace, as follows:

Result

Identification of the NGP in Kallima and closely related butterflies

Using Remane’s criteria [45], a rigorous comparative morphological method, we identified that the K. inachus leaf pattern was composed of the elements described by the NGP (Figure 1e). Our analysis decomposed the main vein of the leaf pattern into three NGP elements: the upper side of the border proximal (BOp) element, the lower side of the central distal (Cd) element in the forewing, and the Cd element in the hindwing. The analysis then decomposed the left-sided lateral vein pattern into six NGP elements: upper side of the Cd element, discal spots (DS) and central proximal (Cp) element (closely attached to form a single straight line), basal (B) and root (R) elements in the forewing, and the Cp element (fragmented into two elements) in the hindwing. The right-sided lateral vein pattern was composed of two NGP elements: the BOp and border distal (BOd) element in the hind wing. The eyespots (ESs) in both the fore and hind wings became vestigial. In this analysis, our rigorous method validated the inference proposed by Süffert [22]. We also examined Kallima paralekta and revealed that the leaf pattern in this species showed almost identical characteristics to those of K. inachus (Figure 2mm). The Kallima leaf patterns were thus depicted by an assembly of the NGP elements.

Subsequently, we found that the wing patterns of 45 butterfly species closely related to genus Kallima were also composed of the NGP elements, although their appearances differed from the leaf-like patterns found in Kallima (Figure 2). Notably, differences among the species could be attributed to differences in geometric shape of the NGP elements. For example, in Kallima, the Cd element in the hindwing formed a straight line (Figure 1e and Figure 2 mm), whereas in Polygonia c-album it did not (Figure 2p). Collectively, the Cd elements in hindwings formed straight lines in twelve species (summarized in Additional file 1: Table S5, character (Ch) 10; Figure 2). These results strongly suggest that the Kallima leaf patterns originated through evolutionary changes in character states of NGP elements from the ancestral species.

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.

Evolutionary accumulation of character state changes in evolution of wing patterns

The above analyses revealed that leaf mimicry evolved via stepwise transitions from one intermediate state to another. Evolution by natural selection is expected to progress through the stepwise accumulation of changes. To gain an improved understanding of the evolution of leaf patterns, we further examined the process of accumulation of character state changes of NGP elements, focusing on mutual and temporal dependency (i.e. contingency) of the changes.

Our Bayesian phylogenetic analyses inferred that evolutionary changes in some pairs of character states were interdependent (Figure 4). Among all possible combinations of pairs of characters (n =55 combination), 19 pairs of character state changes were significantly supported as dependent (logarithmic Bayes Factor (log-BF) >2; Additional file 1: Table S6); these dependencies were summarized as a linkage map (Figure 4b). Subsequent analyses examined whether cases of dependent evolution consisted of mutual or temporal dependence (Additional file 1: Table S8). Among the 19 pairs of character states showing dependent evolution, six demonstrated mutual dependence and 13 evidenced temporal dependence (Z-score >70%; Additional file 1: Table S7 and Figure S4). The temporal dependence indicated that some state changes in NGP elements were contingent upon the background states of other elements, suggesting a temporal order of accumulation of the changes, summarized as a form of network (Figure 4c). The accumulation of character state changes occurred in the following order: (1) independent loss of the hindwing DS and B (Ch 7 and 9); (2) evolution of the hindwing Bd as a straight line (Ch 10) and of the forewing Cd into a single broken straight line (Ch 3); (3) evolution of the upper BOp into a straight pattern (Ch 5); (4) transition of the bend in the hindwing BOp from proximal to distal (Ch 4). On the other hand, after the Cd in the hindwing straightened, the ESs in both fore- and hindwings became vestigial (Ch 6 and 11). Taken together, evolution of the Kallima leaf pattern progressed in concert with the sequential accumulation of state changes in NGP elements.

So, not only did the authors reconstruct the changes required to produce the Kallima wing pattern from an ancestral, generic NGP, but also determined the time order in which these changes occurred. I'd say this is a pretty powerful result.

And finally, the discussion:

Discussion

This study delivers the first clear picture of the evolution of leaf mimicry in Kallima butterflies. Our analyses clarified the ways in which butterfly wing patterns evolve to resemble leaves. From the perspective of comparative morphology, the Kallima leaf patterns are decomposed into homologous pattern elements shared across closely related species. A lineage of leaf mimicry patterns evolved through stepwise changes and intermediate wing patterns. These results were further corroborated by in-depth analyses of trait evolution that revealed contingency in the sequential accumulation of traits; changes in states of elements influenced the subsequent evolution of other state changes. Although some have argued that the gradual evolution of leaf mimicry is improbable [18],[19], our analyses reveal that leaf mimicry evolved gradually from the non-mimetic pattern without a sudden transition. We conclude that Kallima leaf patterns evolved in a stepwise manner through intermediate forms with sequential accumulation of state changes (Figure 5).

Bingo. Task completed.

Furthermore, we have this:

The temporal order of the accumulation of character state changes was assessed using Z-scores. Thus, contingency was a quantitative value and subsequent evolution was influenced to some degree by unique, chance historical events [28],[32],[33]. In the network of contingent evolution, only four character pairs showed strong evidence of contingent evolution, where the occurrence of one state change was highly contingent upon the background state of the other (Z-score >95%; Figure 4c, Additional file 1: Figure S4). The other three pairs of character state changes showed moderate contingency (Z-score >90%). Under the loosest criteria (Z-score >70%), six pairs showed weak contingency, where one state change loosely restricted subsequent state changes. These results strongly suggested that the evolutionary trajectories in this lineage were not completely restricted. In fact, an alternative temporal order in state changes was likely to have occurred. For example, before the loss of the hindwing B (Ch 7), a straight hindwing Bd (Ch 10) occurred in five species (Doleschallia bisaltide, Protogoniomorpha anacardii, Protogoniomorpha parhassus, Yoma Sabina, and Precis archesia). Although the evolutionary restriction varied from weak to strong, our analyses provided evidence that evolution of the leaf pattern resulted in sequential accumulation of character state changes.

So, not only did the authors determine that the leaf mimicry pattern of Kallima butterflies occur in a stepwise manner, with well-defined state transitions of pattern elements in a specific temporal order, but they also used that analysis to determine the emergence of patterns in other, non-mimetic butterflies used in the analysis.

Furthermore, seemingly in anticipation of my own caveats about treating naive views of imperfect mimicry in an injudicious manner, the authors provide this:

We provide the analytical framework for evolution of complex adaptive phenotypes within a phylogenetic context. Comparative morphological analysis revealed that Kallima spp. and closely related species were explained by the same NGP scheme. Thus, the morphological differences between species were attributable to differences in the states of subordinate elements. We suggest that this analytical approach is applicable to the evolution of other types of camouflage (e.g. lichen and tree-bark mimetic patterns). Previous studies demonstrated the utility of phylogenetic approaches to studying the evolution of camouflage and mimicry [56]-[58], but the advantage of the ground plan scheme was unexplored. The phylogenetic homology-based comparative method used here provides a powerful way to explore evolutionary origins and processes of camouflage and mimicry as well as other morphological evolution, although the extent to which this framework can be applied to other cases remains to be determined.

Although our data strongly suggest that leaf mimicry emerged gradually through intermediate states of wing patterns, the survival mechanisms of butterflies with intermediate patterns remain unclear. To date, many believe that intermediate wing patterns represent poor forms of mimicry, which generates criticism of the possibility of gradual evolution of leaf mimicry [18],[19],[59],[60]. One plausible explanation for survival of poor mimics is found in the concept of imperfect mimicry [61], which maintains that the survival of poor mimics (e.g. hoverflies that are poor wasp mimics [62],[63]) is often explained by a trade-off in predator foraging behaviours (e.g. a trade-off between the speed and accuracy of decision-making [64]). This concept suggests the following evolutionary scenario: the larger the area in which a predator seeks prey, the less time the predator has to discriminate whether an object it encounters is edible, the lower the accuracy of discrimination, and the higher the probability that a poor mimic escapes predation [65]. Applying this scenario to poor leaf mimics, predators may misidentify prey as leaves. Because special resemblance of animals to natural objects is termed masquerade, we propose a hypothesis of “imperfect masquerade” for the above phenomena.

Ah, beautiful. Don't you agree?

The authors close their discussion with:

Leaf mimicry provides a textbook example of adaptation and is also a historically contentious subject that has spurred criticism of modern evolutionary synthesis. Although Darwin and Wallace (and subsequent evolutionary biologists) argued for the gradual origin of leaf mimesis, the lack of direct experimental evidence has allowed antagonists to produce alternative evolutionary scenarios (e.g. saltation) for leaf mimesis [19]. In the past, leaf mimicry in Kallima butterfly species was a cornerstone in arguments for saltatory evolution, in which context Goldschmidt argued that this mimicry must have originated suddenly as a ‘hopeful monster’, without any intermediate forms [19]. The discovery revealed in this study refutes such claims and demonstrates that the appearance of leaf mimicry in Kallima spp. has arisen in a stepwise and contingent fashion.

The authors thus conclude:

This study delivers the first clear picture of the evolutionary emergence of leaf mimicry in Kallima butterflies. The evolutionary emergence of leaf mimicry has been a historically contentious issue and remains an unresolved conundrum. Our analyses resolved this conundrum by demonstrating that the leaf pattern evolved gradually from a non-mimetic pattern. Although we could not show the survival mechanisms of butterflies with intermediate patterns, the results of this study strongly suggest evolutionary trajectories toward leaf mimicry via intermediate states of wing patterns, and we therefore proposed an `imperfect masquerade’ to explain the presence of wing patterns with intermediate states. In the future, it will be necessary to investigate how butterflies with such intermediate patterns can survive by investigating the foraging behaviour of predators and escape strategy of butterflies.

In addition, we elaborated a powerful method to explore the evolutionary process of complex adaptive phenotypes. To date, comparative morphological approaches were used to investigate macro-level evolution; however, these approaches could be hardly applied to micro-level evolution, partly because of the lack of appropriate statistical methods to detect subtle phenotypic changes. The method that we developed is based on a comparative morphological approach in combination with phylogenetic Bayesian statistics, which can be applied to various examples of phenotypic evolution such as the evolution of vertebrate neuro-musculo-skeletal systems or that of insect camouflage and mimicry.

This is how it's done. Science, it works. Bitches. :D

Now of course, all that is needed to solidify this into a pretty much impregnable edifice, is tracking of the genes in the requisite species, and that work is underway as I write this. It merely remains for me to hope that my proofreading has eliminated errors before I post this three hours or work. :)

Can you Boil it down for us calisesia [sic] w out gobs of text?

Didn't you read any of my explanatory paragraphs between the quoted sections of the paper, which were provided specifically for that purpose?

What might be noteworthy is.....
“However, the process by which leaf patterns evolved remains unclear.”

So despite me providing not only a full exposition of the paper, but explicit warnings against quote mining, you launch into as blatant and banal a quote mine as only a creationist could. Congratulations for demonstrating that my measures in this vein were justified.

What part of the authors' conclusions at the end that I emphasised in boldface did you not read when performing your cheap and duplicitous quote mine? Or, for that matter, what part of my exposition of the modus operandi of scientific paper writing at the beginning did you also not read? The exposition where I explicitly stated, that the format consists of "here's a problem to be addressed, here's the work we did to address it, and here's our conclusions arising from that work?"

A reminder of one the bits you skipped during your quote mining:

This study delivers the first clear picture of the evolutionary emergence of leaf mimicry in Kallima butterflies. The evolutionary emergence of leaf mimicry has been a historically contentious issue and remains an unresolved conundrum. Our analyses resolved this conundrum by demonstrating that the leaf pattern evolved gradually from a non-mimetic pattern.

This is as unambiguous a statement of success of the endeavour as anyone reading the paper honestly could wish for. I conclude by your pathetic response above, that you are neither interested in honest discourse, nor honest appraisal of scientific research, but instead are intent solely on propagandising for your sad little doctrine. But I didn't provide this exposition for your benefit, because I've had over a decade's worth of experience elsewhere of creationist mendacity, and therefore had no expectation that you would respond with anything other than a manifestly duplicitous evasion of the data. Others here who have already benefited from the exposition I've provided, will no doubt subject your sleazy little pseudo-response to the contempt it manifestly deserves.

Can you address the fact that You are up against a random numbers game no matter how many experiments and observations you conduct.

Translation: "I don't care how much data destroys my assertions, I'm going to keep peddling them", which is all you're offering here.

What part of "the experiments in question demonstrate that this happened" do you not understand?

Though once again, the fact that scientists and mathematicians have placed what you snidely dismiss as "a random numbers game" on a rigorous footing, is another of those inconvenient facts you'll squirm to avoid at all costs, because that diligent effort on their part destroys your infantile posturings.

Furthermore, when mechanisms exist allowing randomly emerging new features to persist, which is clearly and demonstrably the case from thousands of examples in the biosphere, your apologetics simply look even more underhand and dishonest than before. But I don't expect creationists to be anything else, not least because they have to keep lying to themselves in order to continue propping up their worthless, evidence-free, assertion-laden doctrine. Quite simply, all you have to offer here is fatuous stonewalling coupled to blatant fabrication, and you're fooling no one with your egregious abuse of proper discourse.

Heard of the Ninth Commandment, have you? I suggest you apply it sometime, given that you're such an enthusiast for the mythology containing it.