Evolutionary dependence of host type and chasmothecial appendage morphology in obligate plant parasites belonging to Erysipheae (powdery mildew, Erysiphaceae).

Evolutionary relationships between the morphological and ecological traits of fungi are poorly understood. The appendages of chasmothecia, which are sexual reproductive organs of Erysiphaceae, are considered to play a crucial role in the overwintering strategies of these fungi on host plants. Previous studies suggested that both the host type and appendage morphology evolved at the same nodes and transitioned from complex appendages on deciduous hosts to simple appendages on herb/evergreen hosts. However, the evolutionary dependence between host type and appendage morphology remains unproven owing to the limited species data used in analyses. To elucidate the evolutionary relationship between host type and appendage morphology, we used phylogenetic comparative methods (PCMs) to investigate the state transition, ancestral state, evolutionary dependence, and contingent evolution within Erysipheae, the largest and most diverse tribe in Erysiphaceae. Our PCMs, based on a comprehensive data set of Erysipheae, revealed that the most ancestral states were deciduous host types and complex appendages. From these ancestral states, convergent evolution toward the herb/evergreen host types and simple appendages occurred multiple times at the same nodes. For the first time in Erysiphaceae, we detected an evolutionary dependence between host type and appendage morphology. This is one of the few examples in which evolutionary dependence between host phenology and morphological traits in plant-parasitic fungi was demonstrated using PCMs. Appendage simplification on herb/evergreen hosts and complications on deciduous hosts can be reasonably explained by the functional advantages of each appendage type in different overwintering strategies. These expected appendage functions can explain approximately 90% of host type and appendage morphology combinations observed in the analyzed taxa. However, our results also highlighted the occurrence of evolutionary shifts that deviate from the expected advantages of each appendage morphology. These seemingly irrational shifts might be interpretable from the flexibility of overwintering strategies and quantification of appendage functions.

Lifestyle Evolution Analysis by Binary-State Speciation and Extinction (BiSSE) Model.

Phylogenetic comparative methods (PCMs) combine statistics and evolutionary models to infer the dynamics of trait evolution and diversification that underlie the observed phylogeny. While PCMs have been used to study macro-evolutionary processes and evolutionary transitions of macroorganisms, their application to microbes is still limited. With the abundance of publicly available genomic and trait character data for diverse microbes nowadays, applications of PCMs on these data can provide insights into the fundamental principles that govern microbial evolution. Here, we introduce the Binary-State Speciation and Extinction (BiSSE) model, which is a relatively simple yet powerful approach for analyzing trait evolution. We begin by explaining the theoretical background and intuition behind the BiSSE model. Then, R commands for running the BiSSE model are presented. Finally, we introduce a case study that successfully applied the BiSSE model to investigate generalist and specialist microbial lifestyle evolution.

Phenotypic systems biology for organisms: Concepts, methods and case studies.

Design principles of phenotypes in organisms are fundamental issues in physical biology. So far, understanding "systems" of living organisms have been chiefly promoted by understanding the underlying biomolecules such as genes and proteins, and their intra- and inter-relationships and regulations. After a long period of sophistication, biophysics and molecular biology have established a general framework for understanding 'molecular systems' in organisms without regard to species, so that the findings of fly studies can be applied to mouse studies. However, little attention has been paid to exploring "phenotypic systems" in organisms, and thus its general framework remains poorly understood. Here I review concepts, methods, and case studies using butterfly and moth wing patterns to explore phenotypes as systems. First, I present a unifying framework for phenotypic traits as systems, termed multi-component systems. Second, I describe how to define components of phenotypic systems, and also show how to quantify interactions among phenotypic parts. Subsequently, I introduce the concept of the macro-evolutionary process, which illustrates how to generate complex traits. In this point, I also introduce mathematical methods, "phylogenetic comparative methods", which provide stochastic processes along molecular phylogeny as bifurcated paths to quantify trait evolution. Finally, I would like to propose two key concepts, macro-evolutionary pathways and genotype-phenotype loop (GP loop), which must be needed for the next directions. I hope these efforts on phenotypic biology will become one major target in biophysics and create the next generations of textbooks. This review article is an extended version of the Japanese article, Biological Physics in Phenotypic Systems of Living Organisms, published in SEIBUTSU-BUTSURI Vol. 61, p. 31-35 (2021).

Evolutionary patterns of host type and chasmothecial appendage morphology in obligate plant parasites belonging to Cystotheceae (powdery mildew, Erysiphaceae).

The chasmothecial appendages of Erysiphaceae are considered to function in the overwintering strategy and evolve morphologically in line with transitions of different host type. However, the evolutionary patterns and relationships of these traits have not yet been verified using statistical models based on phylogenetic information. We aimed to clarify the evolutionary process of host type and appendage morphology in Cystotheceae using phylogenetic comparative methods (PCMs) and to evaluate the evolutionary relationship of these traits. The ancestral state estimation of host types showed that the deciduous type is the most ancestral in Cystotheceae, and the herb or evergreen types evolved secondarily four times and twice, respectively. Branched- or circinate-type appendages were estimated to be the most ancestral, and the mycelioid and rudimentary types evolved secondarily thrice and once, respectively. The results of the random forest analysis showed that the host type was predictable from the phylogeny and appendage morphology. The ancestral state estimation suggested that simultaneous transitions of the host type and appendage morphology occurred at several ancestral nodes. These results suggest some functional relationships between host type and appendage morphology, but there was no statistical support for an overall trend in evolutionary dependence between these traits. Our results demonstrate the utility of PCMs in the study of trait evolution in Cystotheceae, which can be applied to a broader phylogeny of powdery mildews to elucidate the evolutionary relationship and functional causality of phenotypic traits.

Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera.

Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.

Multicomponent structures in camouflage and mimicry in butterfly wing patterns.

Understanding how morphological structures are built is essential for appreciating the morphological complexity and divergence of organisms. One representative case of morphological structures is the camouflage and mimicry of butterfly wing patterns. Some previous studies have questioned whether camouflage and mimicry are truly structures, considering that they rely on coloration. Nevertheless, our recent study revealed that the leaf pattern of Kallima inachus butterfly wings evolved through the combination of changes in several pigment components in a block-wise manner; it remains unclear whether such block-wise structures are common in other cases of camouflage and mimicry in butterflies and how they come about. Previous studies focused solely on a set of homologous components, termed the nymphalid ground plan. In the present study, we extended the scope of the description of components by including not only the nymphalid ground plan but also other common components (i.e., ripple patterns, dependent patterns, and color fields). This extension allowed us to analyze the combinatorial building logic of structures and examine multicomponent structures of camouflage and mimicry in butterfly wing patterns. We investigated various patterns of camouflage and mimicry (e.g., masquerade, crypsis, Müllerian mimicry, Batesian mimicry) in nine species and decomposed them into an assembly of multiple components. These structural component analyses suggested that camouflage and mimicry in butterfly wing patterns are built up by combining multiple types of components. We also investigated associations between components and the kinds of camouflage and mimicry. Several components are statistically more often used to produce specific types of camouflage or mimicry. Thus, our work provides empirical evidence that camouflage and mimicry patterns of butterfly wings are mosaic structures, opening up a new avenue of studying camouflage, and mimicry from a structural perspective.

On the Origin of Complex Adaptive Traits: Progress Since the Darwin Versus Mivart Debate.

The evolutionary origin of complex adaptive traits has been a controversial topic in the history of evolutionary biology. Although Darwin argued for the gradual origins of complex adaptive traits within the theory of natural selection, Mivart insisted that natural selection could not account for the incipient stages of complex traits. The debate starting from Darwin and Mivart eventually engendered two opposite views: gradualism and saltationism. Although this has been a long-standing debate, the issue remains unresolved. However, recent studies have interrogated classic examples of complex traits, such as the asymmetrical eyes of flatfishes and leaf mimicry of butterfly wings, whose origins were debated by Darwin and Mivart. Here, I review recent findings as a starting point to provide a modern picture of the evolution of complex adaptive traits. First, I summarize the empirical evidence that unveils the evolutionary steps toward complex traits. I then argue that the evolution of complex traits could be understood within the concept of "reducible complexity." Through these discussions, I propose a conceptual framework for the formation of complex traits, named as reducible-composable multicomponent systems, that satisfy two major characteristics: reducibility into a sum of subcomponents and composability to construct traits from various additional and combinatorial arrangements of the subcomponents. This conceptual framework provides an analytical foundation for exploring evolutionary pathways to build up complex traits. This review provides certain essential avenues for deciphering the origin of complex adaptive traits.

Gradual and contingent evolutionary emergence of leaf mimicry in butterfly wing patterns.

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.

Identification of a novel strong and ubiquitous promoter/enhancer in the silkworm Bombyx mori.

Transgenic techniques offer a valuable tool for determining gene functions. Although various promoters are available for use in gene overexpression, gene knockdown, and identification of transgenic individuals, there is nevertheless a lack of versatile promoters for such studies, and this dearth acts as a bottleneck, especially with regard to nonmodel organisms. Here, we succeeded in identifying a novel strong and ubiquitous promoter/enhancer in the silkworm. We identified a unique silkworm strain whose reporter gene showed strong and ubiquitous expression during the establishment of enhancer trap strains. In this strain, the transposon was inserted into the 5'UTR of hsp90, a housekeeping gene that is abundantly expressed in a range of tissues. To determine whether the promoter/enhancer of hsp90 could be used to induce strong gene expression, a 2.9-kb upstream genomic fragment of hsp90 was isolated (hsp90(P2.9k)), and its transcriptional activation activity was examined. Strikingly, hsp90(P2.9k) induced strong gene expression in silkworm cell cultures and also strongly induced gene expression in various tissues and developmental stages of the silkworm. hsp90(P2.9k) also exhibited significant promoter/enhancer activity in Sf9, a cell culture from the armyworm, suggesting that this fragment might possibly be used as a gene expression tool in other Lepidoptera. We further found that 2.0 kb of hsp90(P2.9k) is sufficient for the induction of strong gene expression. We believe that this element will be of value for a range of studies such as targeted gene overexpression, gene knockdown and marker gene expression, not only in the silkworm but also in other insect species.

Modularity of a leaf moth-wing pattern and a versatile characteristic of the wing-pattern ground plan.

BACKGROUND: One of the most intriguing questions in evolutionary developmental biology is how an insect acquires a mimicry pattern within its body parts. A striking example of pattern mimicry is found in the pattern diversity of moth and butterfly wings, which is thought to evolve from preexisting elements illustrated by the nymphalid ground plan (NGP). Previous studies demonstrated that individuality of the NGP facilitates the decoupling of associated common elements, leading to divergence. In contrast, recent studies on the concept of modularity have argued the importance of a combination of coupling and decoupling of the constituent elements. Here, we examine the modularity of a mimicry wing pattern in a moth and explore an evolvable characteristic of the NGP. RESULTS: This study examined the wings of the noctuid moth Oraesia excavata, which closely resemble leaves with a leaf venation pattern. Based on a comparative morphological procedure, we found that this leaf pattern was formed by the NGP common elements. Using geometric morphometrics combined with network analysis, we found that each of the modules in the leaf pattern integrates the constituent components of the leaf venation pattern (i.e., the main and lateral veins). Moreover, the detected modules were established by coupling different common elements and decoupling even a single element into different modules. The modules of the O. excavata wing pattern were associated with leaf mimicry, not with the individuality of the NGP common elements. For comparison, we also investigated the modularity of a nonmimetic pattern in the noctuid moth Thyas juno. Quantitative analysis demonstrated that the modules of the T. juno wing pattern regularly corresponded to the individuality of the NGP common elements, unlike those in the O. excavata wing pattern. CONCLUSIONS: This study provides the first evidence for modularity in a leaf mimicry pattern. The results suggest that the evolution of this pattern involves coupling and decoupling processes to originate these modules, free from the individuality of the NGP system. We propose that this evolution has been facilitated by a versatile characteristic of the NGP, allowing the association of freely modifiable subordinate common elements to make modules.