A release from developmental bias accelerates morphological diversification in butterfly eyespots.

Development can bias the independent evolution of traits sharing ontogenetic pathways, making certain evolutionary changes less likely. The eyespots commonly found on butterfly wings each have concentric rings of differing colors, and these serially repeated pattern elements have been a focus for evo-devo research. In the butterfly family Nymphalidae, eyespots have been shown to function in startling or deflecting predators and to be involved in sexual selection. Previous work on a model species of Mycalesina butterfly, Bicyclus anynana, has provided insights into the developmental control of the size and color composition of individual eyespots. Experimental evolution has also shown that the relative size of a pair of eyespots on the same wing surface is highly flexible, whereas they are resistant to diverging in color composition, presumably due to the underlying shared developmental process. This fixed color composition has been considered as a prime example of developmental bias with significant consequences for wing pattern evolution. Here, we test this proposal by surveying eyespots across the whole subtribe of Mycalesina butterflies and demonstrate that developmental bias shapes evolutionary diversification except in the genus Heteropsis which has gained independent control of eyespot color composition. Experimental manipulations of pupal wings reveal that the bias has been released through a novel regional response of the wing tissue to a conserved patterning signal. Our study demonstrates that development can bias the evolutionary independence of traits, but it also shows how bias can be released through developmental innovations, thus, allowing rapid morphological change, facilitating evolutionary diversification.

Conserved developmental processes and the formation of evolutionary novelties: examples from butterfly wings.

The origin and diversification of evolutionary novelties-lineage-specific traits of new adaptive value-is one of the key issues in evolutionary developmental biology. However, comparative analysis of the genetic and developmental bases of such traits can be difficult when they have no obvious homologue in model organisms. The finding that the evolution of morphological novelties often involves the recruitment of pre-existing genes and/or gene networks offers the potential to overcome this challenge. Knowledge about shared developmental processes obtained from extensive studies in model organisms can then be used to understand the origin and diversification of lineage-specific structures. Here, we illustrate this approach in relation to eyespots on the wings of Bicyclus anynana butterflies. A number of spontaneous mutations isolated in the laboratory affect eyespots, lepidopteran-specific features, and also processes that are shared by most insects. We discuss how eyespot mutants with disturbed embryonic development may help elucidate the genetic pathways involved in eyespot formation, and how venation mutants with altered eyespot patterns might shed light on mechanisms of eyespot development.

Developmental and genetic mechanisms for evolutionary diversification of serial repeats: eyespot size in Bicyclus anynana butterflies.

Serially repeated pattern elements on butterfly wings offer the opportunity for integrating genetic, developmental, and functional aspects towards understanding morphological diversification and the evolution of individuality. We use captive populations of Bicyclus anynana butterflies, an emerging model in evolutionary developmental biology, to explore the genetic and developmental basis of compartmentalized changes in eyespot patterns. There is much variation for different aspects of eyespot morphology, and knowledge about the genetic pathways and developmental processes involved in eyespot formation. Also, despite the strong correlations across all eyespots in one butterfly, B. anynana shows great potential for independent changes in the size of individual eyespots. It is, however, unclear to what extent the genetic and developmental processes underlying eyespot formation change in a localized manner to enable such individualization. We use micromanipulations of developing wings to dissect the contribution of different components of eyespot development to quantitative differences in eyespot size on one wing surface. Reciprocal transplants of presumptive eyespot foci between artificial selection lines and controls suggest that while localized antagonistic changes in eyespot size rely mostly on localized changes in focal signal strength, concerted changes depend greatly on epidermal response sensitivities. This potentially reflects differences between the signal-response components of eyespot formation in the degrees of compartmentalization and/or the temporal pattern of selection. We also report on the phenotypic analysis of a number of mutant stocks demonstrating how single alleles can affect different eyespots in concert or independently, and thus contribute to the individualization of serially repeated traits.

Pattern formation: a focus on notch in butterfly eyespots.

New observations of early and dynamic expression of Notch in developing lepidopteran wings suggests that this signalling pathway may function in defining the central focus that will specify the butterfly eyespot colour pattern.

Evolution: have wings come, gone and come again?

Can complex traits be re-evolved by lineages that have lost them? Phylogenetic study now suggests that wings may indeed have reappeared several times within the ancestrally wingless stick insects.

BUTTERFLY EYESPOTS: THE GENETICS AND DEVELOPMENT OF THE COLOR RINGS.

The butterfly Bicyclus anynana has a series of distal eyespots on its wings. Each eyespot is composed of a white pupil, a black disc, and a gold outer ring. We applied artificial selection to the large dorsal eyespot on the forewing to produce a line with the gold ring reduced or absent (BLACK) and another line with a reduced black disc and a broad gold ring (GOLD). High heritabilities, coupled with a rapid response to selection, produced two lines of butterflies with very different phenotypes. Other eyespots showed a correlated change in the proportion of their color rings. Surgical experiments were performed on pupal wings from the different lines at the time of eyespot pattern specification. They showed that the additive genetic variance for this trait was in the response of the wing epidermis to signaling from the organizing cells at the eyespot center (the focus). This response was found to vary across different regions of the wing and also between the sexes. The particular eyespot color composition found for each sex, as well as the maintenance of the high genetic variation, are discussed with reference to the ecology of the butterfly, sexual selection, and visual selection by predators.

THERMAL EVOLUTION OF EGG SIZE IN DROSOPHILA MELANOGASTER.

We measured the size of eggs produced by populations of Drosophila melanogaster that had been collected along latitudinal gradients in different continents or that had undergone several years of culture at different temperatures in the laboratory. Australian and South American populations from higher latitudes produced larger eggs when all were compared at a standard temperature. Laboratory populations that had been evolving at 16.5°C produced larger eggs than populations that had evolved at 25°C or 29°C, suggesting that temperature may be an important selective agent in producing the latitudinal clines. Flies from laboratory populations produced larger eggs at an experimental temperature of 16.5°C than at 25°C, and there was no indication of genotype-environment interaction for egg size. Evolution of egg size in response to temperature cannot be accounted for by differences in adult body size between populations. It is not clear which life-history traits are direct targets of thermal selection and which are showing correlated responses, and disentangling these is a task for the future.

RAPID LABORATORY EVOLUTION OF ADULT LIFE-HISTORY TRAITS IN DROSOPHILA MELANOGASTER IN RESPONSE TO TEMPERATURE.

Three replicate lines of Drosophila melanogaster were cultured at each of two temperatures (16.5°C and 25°C) in population cages for 4 yr. The lifespans of both sexes and the fecundity and fertility of the females were then measured at both experimental temperatures. The characters showed evidence of adaptation; flies of both sexes from each selection regime showed higher longevity, and females showed higher fecundity and fertility, than flies from the other selection regime when they were tested at the experimental temperature at which they had evolved. Calculation of intrinsic rates of increase under different assumptions about the rate of population increase showed that the difference between the lines from the two selection regimes became less the higher the rate of population increase, because the lines were more similar in early adulthood than they were later. Despite the increased adaptation of the low-temperature lines to the low temperature, like the high temperature lines they produced progeny at a higher rate at the higher temperature. The lines may have independently evolved adaptations to their respective thermal regimes during the experiment, or there may have been a trade-off between adaptation to the two temperatures, or mutation pressure may have lowered adaptation to the temperature that the flies no longer encountered.

EVOLUTION AND DEVELOPMENT OF BODY SIZE AND CELL SIZE IN DROSOPHILA MELANOGASTER IN RESPONSE TO TEMPERATURE.

We examined the evolutionary and developmental responses of body size to temperature in Drosophila melanogaster, using replicated lines of flies that had been allowed to evolve for 5 yr at 25°C or at 16.5°C. Development and evolution at the lower temperature both resulted in higher thorax length and wing area. The evolutionary effect of temperature on wing area was entirely a consequence of an increase in cell area. The developmental response was mainly attributable to an increase in cell area, with a small effect on cell number in males. Given its similarity to the evolutionary response, the increase in body size and cell size resulting from development at low temperature may be a case of adaptive phenotypic plasticity. The pattern of plasticity did not evolve in response to temperature for any of the traits. The selective advantage of the evolutionary and developmental responses to temperature is obscure and remains a major challenge for future work.

THE EVOLUTIONARY GENETICS AND DEVELOPMENTAL BASIS OF WING PATTERN VARIATION IN THE BUTTERFLY BICYCLUS ANYNANA.

We have studied interactions between developmental processes and genetic variation for the eyespot color pattern on the adult dorsal forewing of the nymphalid butterfly, Bicyclus anynana. Truncation selection was applied in both an upward and a downward direction to the size of a single eyespot consisting of rings with wing scales of differing color pigments. High heritabilities resulted in rapid responses to selection yielding divergent lines with very large or very small eyespots. Strong correlated responses occurred in most of the other eyespots on each wing surface. The cells at the center of a presumptive eyespot (the "focus") act in the early pupal stage to establish the adult wing pattern. The developmental fate of the scale cells within an eyespot is specified by the "signaling" properties of the focus and the "response" thresholds of the epidermis. The individual eyespots can be envisaged as developmental homologues. Grafting experiments performed with the eyespot foci of the selected lines showed that additive genetic variance exists for both the response and, in particular, the signaling components of the developmental system. The results are discussed in the context of how constraints on the evolution of this wing pattern may be related to the developmental organization.

Pattern regulation during the development of the dorsal abdomen in the flesh fly, Sarcophaga agryostoma.

In dipteran flies the adult abdominal epidermis is formed from small nests of diploid histoblast cells which spread out and replace the larval epidermis during metamorphosis. The pattern of nest outgrowth and fusion in Sarcophaga shows that the large dorsal hemitergite is normally formed by the two dorsal nests, the spiracle nest and part of the ventral nest (which also forms the hemisternite). By rotating the dorsal histoblast nests, we demonstrate that the adult segment border lies between the flexible 'intersegmental membrane' (ISM) and the naked anterior strip of tergite, the acrotergite. Deletion of histoblast nests often results in a corresponding deletion of adult structures, accompanied by enlargement of adjacent structures within the segment and in neighbouring segments. Pattern formation is not strictly coupled to cell division (as in imaginal discs), since the nests remaining after an ablation, in spreading to fill vacant areas, generate more cells and larger structures than normal. Nest deletions can also result in regeneration, with remaining nests forming additional structures in the dorsal-ventral or anterior-posterior axis of the segment. The deletion of strips of anterior and intersegmental larval epidermis without histoblasts results in the formation of double-posterior duplications of the adult hemitergite. Although these operations damage adjacent histoblast nests, several features of the results suggest that the duplications arise from the interaction (after healing) of histoblasts with larval cells which they would not normally encounter, leading to the intercalation of histoblast cells bearing intervening anterior-posterior positional values. A similar process of intercalation may occur in normal development, as the histoblasts spread from their local origins across the larval epidermal sheet, replacing the larval cells to form the entire epidermis of the adult segment.

Leg regeneration in the cockroach,Blattella germanica : I. Regeneration from a congruent tibial graft/host junction.

The interactions occuring between graft and host leg epidermis at a congruent junction (non-rotated, homopleural combination of components cut perpendicular to the proximal-distal axis) were studied at the tibia level in the cockroach,Blattella germanica. Grafts were made between dark (Bl) and light (br) cuticle colour mutants. 1) Precise boundaries could not usually be drawn between Bl and br tissue over areas of bare cuticle but spines, hairs and claws could be identified, providing a good indication of the graft or host origin of regenerated structures. 2) When the graft tarsus remained intact, segmented structures were not regenerated from the junction. Host distal tibia and graft proximal tibia interacted to produce a reversed orientation intercalary regenerate, usually composed mainly of host cells which had become more proximal than their level of origin. 3) When the graft tarsus was amputated (or broken off), nearly 50% of congruent junctions regenerated segmented distal structures, which were classified as "autonomous" or "lateral". Amputation of the graft tarsus acted, not through removal of any inhibition, but by hindering healing of the junction because of the apolysis of graft tibial epidermis. 4) Distal structures regenerated autonomously by host and graft components of the junction were either complete or partial (fused at a common level in the tarsus). 5) Lateral regenerates were of joint origin and usually distally incomplete. They were stable and, when amputated, were regenerated to approximately the same level, in the presence or absence of the graft tarsus. 6) It is concluded that autonomous regeneration occurred from junctions which had totally failed to heal, and that lateral regeneration occurred from an unhealed sector of a junction. Laterals were therefore regenerated from a bilaterally symmetrical, partial circumference. They are compared to other incomplete regenerates found in analogous situations. The relationship between transverse organization and distal incompleteness is obscure. 7) Segmented structures are thus regenerated only in situations where host and graft do not heal and interact (at least initially) over all or part of the circumference of the junction: interaction results in the formation of an unsegmented intercalary regenerate comprising the levels normally lying between host and graft on the proximal-distal axis.