Unveiling the secrets of the rainforest's first camouflage master, the "dead leaf butterfly"

The dead leaf butterfly, commonly known as the "dead leaf butterfly", has beautiful blue velvet-like wings with orange markings. When it stops and folds its wings, the gorgeous wings disappear and turn into the appearance of dead leaves, and can even simulate the midrib, secondary veins, petioles and even the decaying morphology of leaves very realistically. What kind of genetic mechanism regulates this magical evolutionary result? The dead leaf butterfly has received considerable attention in multiple disciplines such as taxonomy and morphology, but research on their origin and the genetic basis of the evolution of their magical mimicry is still relatively lacking. Recently, a study by Zhang Wei's team from the School of Life Sciences of Peking University and the Peking University-Tsinghua Joint Center for Life Sciences responded to the above questions. [Please go to the "Fanpu" official account to watch the video]

Written by | Zhang Wei

Most people’s memories of childhood may include a scene of fluttering butterflies. The gorgeous patterns on the butterfly wings are free and agile, opening a beautiful and diverse window for the first acquaintance with the world.

As a completely metamorphosed insect, butterflies have a close relationship with their host plants during the larval stage and are also sensitive to environmental and climate change. They can be used as indicator species for ecological monitoring and to study biodiversity at multiple scales, so they have accumulated a wealth of ecological data. Looking at more than 18,000 butterfly species, the most impressive thing is the tiny butterfly wings. The structure of this organ seems flat and simple, but it has complex biological functions, such as movement, courtship, defense against enemies, and thermal regulation. The butterfly wings, which are simple in structure but complex in function, are the product of nature, driven by pressures such as natural selection and sexual selection . Therefore, they have attracted the attention of biologists and have become a model for ecological and evolutionary research.

How do we understand the diversity of butterfly wings? Among the many butterfly shadows, which system should be chosen to conduct what kind of research? This is a question from beginning to end. For example, in the Amazon jungle, the poisonous butterfly, which has similar bright wing patterns in different species and has undergone radiation evolution, is an ideal system for studying speciation, interspecific hybridization, and adaptive radiation. In the rainforests of Southeast Asia, the non-toxic swallowtail butterfly presents bright wing patterns that mimic the poisonous swallowtail butterfly, but this mimicry phenomenon is limited to female butterflies and shows polymorphism, which provides inspiration for studying phenotypic diversity, sexual dimorphism (differences such as structure and function between female and male individuals in sexually reproducing organisms) , and adaptive evolution.

This article focuses on a type of secretive butterfly, whose camouflage and imitation of leaves are easily overlooked by humans and their natural enemies. Leaves are everywhere in nature, and this phenomenon of animals imitating plants is very beneficial and is widely found in the animal kingdom, such as the Amazon leaf fish, the Malaysian leaf frog, the leaf cricket, the pseudo-leaf katydid, etc., but it does not prevent the protagonist of this article from becoming one of the most famous and eye-catching leaf-shaped mimics.

Figure 1: The wing phenotype of the Chinese subspecies of the dead leaf butterfly. The ventral side of its wings shows a lifelike leaf-shaped phenotype, and the dorsal side shows bright spots. 丨Source: Teng Dequn

A butterfly with different selective pressures on the ventral side of its wings

Species of the genus Nymphalidae are widely distributed in East and Southeast Asia and are well-known for their distinctive wing patterns. When their wings are folded back, they cleverly simulate the brown withered leaves. The leaf-like pattern is very lifelike, and it consists of elements including the midrib, secondary veins, petioles, and even has a pattern similar to mildew spots ( Figure 1 ). This trait is likely driven by natural selection pressure . When it spreads its wings and flies, it presents a bright pattern on the back of the wings. Some species of the genus Nymphalidae have bright patchy patterns. This trait may be related to courtship and is driven by sexual selection pressure, but it may also be to warn natural enemies. It is another result of natural selection and needs further research and confirmation.

Therefore, the author believes that the asymmetrical pattern on the dorsal and ventral surfaces of its wings well demonstrates the evolvability of phenotypic diversity under conservative developmental constraints, and can be used to study the shaping of phenotype and function by different selection pressures .

Evolutionary biologists' interest in the leaf butterfly was first recorded in Wallace's 1889 book Darwinism: An Inquiry into the Theory of Natural Selection, with Some Applications, which considered it " the most wonderful and undoubted example of protective resemblance among butterflies " [1] . In the more than 100 years since then, scientists have studied it from multiple perspectives, including taxonomy, phylogeny, physiology, and morphology, and believe that its leaf shape phenotype is a product of gradual evolution, laying the foundation for exploring the evolution and genetic mechanisms of various wing shape elements that constitute leaf mimicry.

On August 2, 2022, the author and team members published a research article in the journal Cell, revealing the evolution and genetic mechanism of the genus Nymphalidae and its leaf mimicry. By collecting and analyzing butterfly samples from 20 genera in the Nymphalidae family, it was found that leaf mimicry showed convergent evolution in the Nymphalidae family, that is, multiple butterflies independently evolved pseudo-leaf phenotypes under similar selection pressures. The multiple species of the genus Nymphalidae form a monophyletic group, which means that the leaf mimicry of the genus Nymphalidae may be inherited from their common ancestor.

In the process of collecting samples, the author and his team also collected samples of six species of the genus Nymphalidae in 11 geographical areas in East Asia and Southeast Asia ( Figure 2 ). Although we know that no two leaves are exactly the same, it is still a big challenge to identify the commonalities from individual differences and identify which species they belong to . However, compared with this, the greater difficulty may come from discovering their presence in those densely forested habitats, which is an important prerequisite for conducting in-depth research on them.

Figure 2 Geographical distribution of samples collected from the genus Nymphalidae. Samples of three species were collected in the collection area in the upper left corner of the figure (Medog County), while samples of a single species were collected in other collection locations. Source: Reference [2]

The multidisciplinary team members have obtained very important clues in their scientific investigations year after year, that is, at least three species of the genus Nymphalidae have been found in the rainforest of Medog County, Tibet, which is very different from the situation where only a single species can be collected in any other area. Even so, in the eyes of the author, this Medog rainforest still contains too many unknown biodiversity resources, and there may still be unknown species of the genus Nymphalidae flying among them.

Figure 3 The Guoguotang bend of the Yarlung Zangbo River in Medog County. Photo by Zhang Wei

The eastern Himalayas are the starting point of differentiation

Medog County is located at the eastern end of the Himalayas. The Yarlung Zangbo River, the world's largest river with the highest average riverbed altitude, flows through here, forming the deepest and longest river canyon, the Yarlung Zangbo Grand Canyon, which has opened up a channel for the water vapor barrier between the Qinghai-Tibet Plateau and the Indian Ocean. Its thousands of meters of altitude difference also provides a diverse habitat for the rich local biological species. In the rainy season in Medog, the Yarlung Zangbo River and its tributaries roar along the valley, as if playing a hymn to life, which wipes away the fatigue of the distant visitors who have just crossed the Mila Mountain, Sejila Mountain, and Galungla Mountain. In this secret lotus land, the origin of the genus of dead leaf butterfly is about to be revealed.

Figure 4: About to pass through the Galungla Tunnel to reach Medog. Photo by Zhang Wei

Based on the discovery in Medog, the author and his team proposed the hypothesis that the eastern Himalayas, due to its huge altitude gradient changes, formed a diverse microenvironment and may be the differentiation center of the genus Lepidoptera . However, another possibility is not ruled out, that the region is a refuge for the genus Lepidoptera, allowing it to survive the ice age.

The actual research results confirmed the first hypothesis. The results of phylogenetic, population history, habitat model and other analyses showed that populations of multiple species of the genus Lepidoptera all had a trend of migrating out of the eastern Himalayas and spreading to the islands of Southeast Asia. The eastern Himalayas may have provided a relatively suitable habitat for Lepidoptera in many historical periods, and the time of species differentiation within the genus may also correspond to the uplift period of the Qinghai-Tibet Plateau.

At this point, the dead leaf butterfly originated and differentiated in this magnificent environment of mountains and rivers. Its light butterfly wings have been flying for hundreds of thousands of years, and the starting point of differentiation began in the southeastern Qinghai-Tibet Plateau. At that time, islands such as Hainan and Taiwan were connected to the mainland through land bridges. Java, Sumatra, Borneo and the Thai-Malay Peninsula formed the ancient Sundaland. Through the ancient land bridges of the Ice Age, the dead leaf butterfly genus further migrated and differentiated. The author's team speculates that some of the current unique island species are likely to be part of the previous large population, which was isolated and speciated due to subsequent climate change and sea level rise.

In this torrent of geological upheavals and climate change, the evolution of the genus Nymphalidae reveals the relationship between mountain biota and other lowland biodiversity hotspots, and also provides an important entry point for understanding the formation of species diversity.

Figure 5: On the surging Yarlung Zangbo River (taken near Ani Bridge). Photo by Zhang Wei

To study a species in depth, we must not only understand its habitat, but also its life history. The research team selected the Chinese subspecies of the genus, the leaf butterfly, which became a model (a biological species selected for scientific research to reveal the general laws of life science) just like fruit flies, mice, nematodes, and Arabidopsis thaliana. However, there is no experience to unlock this model, and only multiple attempts and explorations are needed.

The breeding room is located on the second underground floor. It is hot and stuffy, but it provides a constant temperature and humidity environment for the dead leaf butterfly and its host plants. It also once again enabled the author's team to make an unexpected discovery, that is, they found that the dead leaf butterfly has at least 10 discrete leaf shape phenotypes ( Figure 6 ). These 10 phenotypes were further confirmed through family experiments, and it was found that they may be controlled by Mendelian single loci, and it is presumed that there may be 5 alleles.

Figure 6 Ten discrete leaf shape phenotypes were found in the Chinese subspecies of the dead leaf butterfly, which are speculated to be controlled by a Mendelian locus. 丨Image source: Reference [2]

One gene controls diverse leaf-wing patterns

By integrating a variety of genomics and gene editing analysis methods, the research team identified a single gene cortex that is involved in controlling this series of leaf shape phenotypes and has 5 haplotypes. This not only confirms the previous hypothesis, but also gradually discovered that the linkage disequilibrium between its haplotypes is maintained by multiple mechanisms such as chromosome inversion and topological association domains. Corresponding to its regulation of wing phenotypes, this gene exhibits expression characteristics related to the wing development of the dead leaf butterfly, and its mosaic deletion mutant also exhibits mottled wing patterns ( Figure 7 ).

Figure 7 Phenotypes of individuals of the leaf butterfly S. sinensis based on CRISPR/Cas9 gene editing. The chimeric deletion mutant of the cortex gene exhibits a mottled wing phenotype, which means that knocking out this gene may affect the formation of the leaf shape phenotype. Image source: Reference [2]

In fact, this famous cortex gene is not an ordinary one. As a toolbox gene that controls the development of butterfly wings (a gene that participates in the development process of biological morphology and structure) , it has also been found to be involved in controlling the industrial melanization phenotype of birch loopers and the wing pattern of Mussopus mimicry [3], and has already made remarkable achievements. This type of toolbox gene plays an important role in the development process and is easily favored by natural selection pressure, thereby obtaining more regulatory methods and participating in more functions. Taking this as an example, the seemingly contradictory concepts of developmental constraints and evolutionary innovation have been reconciled, which also demonstrates the evolvability in the conservative development process and further reveals the possible generation mechanism of genetic diversity and phenotypic diversity.

So far, the genetic mechanism of leaf mimicry has been explained in the Chinese subspecies of the genus Nymphalidae, and to explore how it originated and evolved in many species of the genus Nymphalidae, we have to continue the unknown exploration. My team found that different leaf phenotypes exist in multiple species of the genus Nymphalidae, and different phenotypes and genotypes have different frequencies in wild populations of multiple species. To maintain such polymorphism, my team hypothesized that it may be under the pressure of natural selection, that is, it has been subjected to long-term balancing selection so that the polymorphism is retained in each species.

If this hypothesis is confirmed, it will be an exciting discovery. For a long time, polymorphism driven by balanced selection pressure has been mostly seen at the subspecies level or between species with a short divergence time. This is due to the fact that if individuals with different phenotypes have different fitness, the chances of passing on the genetic information corresponding to their phenotypes to their offspring will be different. This difference will eventually cause the number of offspring produced by individuals in the population to be lower than the expected level, which puts pressure on the continuation of the population, also known as genetic load.

By typing the cortex genes of multiple species of the genus Nymphalidae and constructing population genetics models, the best-fitting model obtained by our team showed that the leaf mimicry polymorphism of the genus Nymphalidae has undergone balancing selection. The pressure of natural selection has shaped this diverse and exquisite leaf camouflage.

The unveiling of the genus of dead leaf butterfly and its leaf mimicry has come to an end, but the pace of exploration has never stopped, and the brainstorming has just begun. The asymmetry of the dorsal and ventral surfaces of butterfly wings, the scattered structural colors and pigment colors, the transformation of wing patterns in the dry and rainy seasons, the hidden species, the unknown phenotypes... and the adaptive evolution, phenotypic plasticity, individual development and evolutionary innovation, speciation, biodiversity generation... all kinds of life evolution implied under the appearance. From 3.5 billion years ago to the present, all the reservations need to be re-recognized, and all the unknowns need to be further explored. This is another new rainy season. At this moment, among the thousands of mountains and valleys of Motuo, there are rainbows after the rain and birds singing in the empty mountains ( Figure 8 ).

Figure 8: In the Medog rainforest (collected near Dexing Township). Photo by Zhang Wei

And those who chase butterflies will set out again, for the fluttering butterfly shadows, thousands and thousands of times.

References

[1] Wallace, AR (1889). Darwinism: An Exploitation of the Theory of Natural Selection with Some of its Applications. (London: Macmillan).

[2]Wang, S., Teng, D., Li X., Yang, P., Da, W., Zhang, Y., Zhang, Y., Liu, G., Zhang,

[3] Nadeau, NJ, Pardo-Diaz, C., Whibley, A., Supple, MA, Saenko, SV, Wallbank, RW, Wu, GC, Maroja, L., Ferguson, L., Hanly, JJ, et al. (2016). The gene cortex controls mimicry and crypsis in butterflies and moths. Nature 534, 106–110.

Produced by: Science Popularization China

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