Evaluating old truths: Final adult size in holometabolous insects is set by the end of larval development.

For centuries, it has been understood that the final size of adult holometabolous insects is determined by the end of the larval stage, and that once they transform to adults, holometabolous insects do not grow. Despite this, no previous study has directly tested these "old truths" across holometabolous insects. Here, we demonstrate that final adult size is set at the end of the last larval stage in species representing each of the four orders of holometabolous insects: the fruit fly Drosophila melanogaster (Diptera), the tobacco hornworm Manduca sexta (Lepidoptera), the dung beetle Onthophagus taurus (Coleoptera), and the Florida carpenter ant Camponotus floridanus (Hymenoptera). Furthermore, in both D. melanogaster and C. floridanus, we show that the size of adult individuals fluctuates but does not significantly change. Therefore, our study finally confirms these two basic assumptions in the biology of insects, which have for centuries served as the foundation for studies of insect growth, size, and allometry.

Deep conservation and co-option of programmed cell death facilitates evolution of alternative phenotypes at multiple biological levels.

Alternative phenotypes, such as polyphenisms and sexual dimorphisms, are widespread in nature and appear at all levels of biological organization, from genes and cells to morphology and behavior. Yet, our understanding of the mechanisms through which alternative phenotypes develop and how they evolve remains understudied. In this review, we explore the association between alternative phenotypes and programmed cell death, a mechanism responsible for the elimination of superfluous cells during development. We discuss the ancient origins and deep conservation of programmed cell death (its function, forms and underlying core regulatory gene networks), and propose that it was co-opted repeatedly to generate alternative phenotypes at the level of cells, tissues, organs, external morphology, and even individuals. We review several examples from across the tree of life to explore the conditions under which programmed cell death is likely to facilitate the evolution of alternative phenotypes.

My road to the ants: A model clade for eco-evo-devo.

This chapter is the story of how I pioneered ants as a system for studying eco-evo-devo, a field that integrates developmental biology with ecology and evolutionary biology. One aim of eco-evo-devo is to understand how the interactions between genes and their environments during development facilitates the origin and evolution of novel phenotypes. In a series of six parts, I review some of the key discoveries from my lab on how novel worker caste systems in ants--soldiers and supersoldiers--originated and evolved. I also discuss some of the ideas that emerged from these discoveries, including the role that polyphenisms, hidden developmental potentials, and rudimentary organs play in facilitating developmental and evolutionary change. As superorganisms, I argue that ants are uniquely positioned to reveal types of variation that are often difficult to observe in nature. In doing so, they have the potential to transform our view of biology and provide new perspectives in medicine, agriculture, and biodiversity conservation. With my story I hope to inspire the next generation of biologists to continue exploring the unknown regions of phenotypic space to solve some of our most pressing societal challenges.

Warm and arid regions of the world are hotspots of superorganism complexity.

Biologists have long been fascinated by the processes that give rise to phenotypic complexity of organisms, yet whether there exist geographical hotspots of phenotypic complexity remains poorly explored. Phenotypic complexity can be readily observed in ant colonies, which are superorganisms with morphologically differentiated queen and worker castes analogous to the germline and soma of multicellular organisms. Several ant species have evolved 'worker polymorphism', where workers in a single colony show quantifiable differences in size and head-to-body scaling. Here, we use 256 754 occurrence points from 8990 ant species to investigate the geography of worker polymorphism. We show that arid regions of the world are the hotspots of superorganism complexity. Tropical savannahs and deserts, which are typically species-poor relative to tropical or even temperate forests, harbour the highest densities of polymorphic ants. We discuss the possible adaptive advantages that worker polymorphism provides in arid environments. Our work may provide a window into the environmental conditions that promote the emergence of highly complex phenotypes.

Nano-CT imaging of larvae in the ant Pheidole hyatti reveals coordinated growth of a rudimentary organ necessary for soldier development.

The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes-the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small-headed minor workers and larger, big-headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head-to-body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano-CT imaging and three-dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye-antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye-antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter-organ coordination as compared with other insects to generate the big-headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter-organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution.

The origin of wing polyphenism in ants: An eco-evo-devo perspective.

The evolution of eusociality, where solitary individuals integrate into a single colony, is a major transition in individuality. In ants, the origin of eusociality coincided with the origin of a wing polyphenism approximately 160 million years ago, giving rise to colonies with winged queens and wingless workers. As a consequence, both eusociality and wing polyphenism are nearly universal features of all ants. Here, we synthesize fossil, ecological, developmental, and evolutionary data in an attempt to understand the factors that contributed to the origin of wing polyphenism in ants. We propose multiple models and hypotheses to explain how wing polyphenism is orchestrated at multiple levels, from environmental cues to gene networks. Furthermore, we argue that the origin of wing polyphenism enabled the subsequent evolution of morphological diversity across the ants. We finally conclude by outlining several outstanding questions for future work.

Origin and elaboration of a major evolutionary transition in individuality.

Obligate endosymbiosis, in which distantly related species integrate to form a single replicating individual, represents a major evolutionary transition in individuality1-3. Although such transitions are thought to increase biological complexity1,2,4-6, the evolutionary and developmental steps that lead to integration remain poorly understood. Here we show that obligate endosymbiosis between the bacteria Blochmannia and the hyperdiverse ant tribe Camponotini7-11 originated and also elaborated through radical alterations in embryonic development, as compared to other insects. The Hox genes Abdominal A (abdA) and Ultrabithorax (Ubx)-which, in arthropods, normally function to differentiate abdominal and thoracic segments after they form-were rewired to also regulate germline genes early in development. Consequently, the mRNAs and proteins of these Hox genes are expressed maternally and colocalize at a subcellular level with those of germline genes in the germplasm and three novel locations in the freshly laid egg. Blochmannia bacteria then selectively regulate these mRNAs and proteins to make each of these four locations functionally distinct, creating a system of coordinates in the embryo in which each location performs a different function to integrate Blochmannia into the Camponotini. Finally, we show that the capacity to localize mRNAs and proteins to new locations in the embryo evolved before obligate endosymbiosis and was subsequently co-opted by Blochmannia and Camponotini. This pre-existing molecular capacity converged with a pre-existing ecological mutualism12,13 to facilitate both the horizontal transfer10 and developmental integration of Blochmannia into Camponotini. Therefore, the convergence of pre-existing molecular capacities and ecological interactions-as well as the rewiring of highly conserved gene networks-may be a general feature that facilitates the origin and elaboration of major transitions in individuality.

Interruption points in the wing gene regulatory network underlying wing polyphenism evolved independently in male and female morphs in Cardiocondyla ants.

Wing polyphenism in ants, which produces a winged female queen caste and a wingless female worker caste, evolved approximately 150 million years ago and has been key to the remarkable success of ants. Approximately 20 million years ago, the myrmicine ant genus Cardiocondyla evolved an additional wing polyphenism among males producing two male morphs: wingless males that fight to enhance mating success and winged males that disperse. Here we show that interruption of rudimentary wing-disc development in larvae of the ant Cardiocondyla obscurior occurs further downstream in the network in wingless males as compared with wingless female workers. This pattern is corroborated in C. kagutsuchi, a species from a different clade within the genus, indicating that late interruption of wing development in males is conserved across Cardiocondyla. Therefore, our results show that the novel male wing polyphenism was not developmentally constrained by the pre-existing female wing polyphenism and evolved through independent alteration of interruption points in the wing gene network. Furthermore, a comparison of adult morphological characters in C. obscurior reveals that developmental trajectories lead to similar morphological trait integration between winged and wingless females, but dramatically different integration between winged and wingless males. This suggests that the alternative sex-specific developmental routes to achieve winglessness in the genus Cardiocondyla may have evolved through different selection regimes acting on wingless males and females.

The genome of the water strider Gerris buenoi reveals expansions of gene repertoires associated with adaptations to life on the water.

BACKGROUND: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group. RESULTS: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000 Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats. CONCLUSIONS: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders.

Social regulation of a rudimentary organ generates complex worker-caste systems in ants.

The origin of complex worker-caste systems in ants perplexed Darwin1 and has remained an enduring problem for evolutionary and developmental biology2-6. Ants originated approximately 150 million years ago, and produce colonies with winged queen and male castes as well as a wingless worker caste7. In the hyperdiverse genus Pheidole, the wingless worker caste has evolved into two morphologically distinct subcastes-small-headed minor workers and large-headed soldiers8. The wings of queens and males develop from populations of cells in larvae that are called wing imaginal discs7. Although minor workers and soldiers are wingless, vestiges or rudiments of wing imaginal discs appear transiently during soldier development7,9-11. Such rudimentary traits are phylogenetically widespread and are primarily used as evidence of common descent, yet their functional importance remains equivocal1,12-14. Here we show that the growth of rudimentary wing discs is necessary for regulating allometry-disproportionate scaling-between head and body size to generate large-headed soldiers in the genus Pheidole. We also show that Pheidole colonies have evolved the capacity to socially regulate the growth of rudimentary wing discs to control worker subcaste determination, which allows these colonies to maintain the ratio of minor workers to soldiers. Finally, we provide comparative and experimental evidence that suggests that rudimentary wing discs have facilitated the parallel evolution of complex worker-caste systems across the ants. More generally, rudimentary organs may unexpectedly acquire novel regulatory functions during development to facilitate adaptive evolution.

Lack of interruption of the gene network underlying wing polyphenism in an early-branching ant genus.

Ants evolved about 140 million years ago and have diversified into more than 15,000 species with tremendous ecological and morphological diversity, yet evolution of the gene regulatory networks (GRNs) underlying this diversification remains poorly understood. Wing polyphenism, the ability of a single genome to produce either winged or wingless castes during development in response to environmental cues, is a nearly universal feature of ants. The underlying wing GRN is evolutionarily labile in worker castes of phylogenetically derived species: it is conserved in winged castes but interrupted at different points in wingless castes of different species. However, it remains unknown whether the wing GRN is interrupted in wingless castes of species from early branching lineages, and if so, whether it is interrupted at similar locations in worker castes of derived species. We therefore used in situ hybridization to assay the expression of nine genes in the wing GRN in wing imaginal discs of larvae from two species from the early branching ('basal') genus Mystrium. These species possess two castes each: Mystrium rogeri has winged queens and wingless workers, and M. oberthueri has wingless queens and wingless workers. In contrast to derived species, we found no evidence of interruption points in the wing GRN kernel of wingless castes. Our finding supports: (1) a "phylogenetic ladder model" of wing GRN evolution, where interruption points move further upstream in the wing GRN as ant lineages become more derived; and (2) that evolutionary lability of the GRN underlying wing polyphenism originated later during ant evolution.

Early Activation of MAPK and Apoptosis in Nutritive Embryos of Calyptraeid Gastropods.

Investigation of alternative phenotypes, different morphologies produced by a single genome, has contributed novel insights into development and evolution. Yet, the mechanisms underlying developmental switch points between alternative phenotypes remain poorly understood. The calyptraeid snails Crepidula navicella and Calyptraea lichen produce two phenotypes: viable and nutritive embryos, where nutritive embryos arrest their development after gastrulation and are ingested by their viable siblings as a form of intracapsular nutrition. Here, we investigate the activity of mitogen-activated protein kinase (MAPK, ERK1/2) and apoptosis during early cleavage. MAPK and apoptosis, found in a previous transcriptomic study, are known to be involved in organization of other spiralian embryos and nutritive embryo development, respectively. In the model Crepidula fornicata, MAPK activation begins at the 16-cell stage. In contrast, we discovered in C. navicella and C. lichen that many embryos begin MAPK activation at the one-cell stage. A subset of embryos shows a similar pattern of MAPK activation to C. fornicata at later stages. In all stages where MAPK is detected, the activation pattern is highly variable, frequently occurring in all quadrants or in multiple tiers of cells. We also detected apoptosis in cleaving embryos, while C. fornicata and Crepidula lessoni, which do not produce nutritive embryos, show no signs of apoptosis during cleavage. Our results show that MAPK and apoptosis are expressed during early development in species with nutritive embryos, and raises the possibility that these processes may play a role and even interact with one another in producing the nutritive embryo phenotype.

Regulation, development, and evolution of caste ratios in the hyperdiverse ant genus Pheidole.

Ant colonies are considered complex biological systems because many individuals are divided into different castes that interact to efficiently perform their tasks. Colonies in the hyperdiverse ant genus Pheidole have evolved a worker caste with at least two subcastes: soldiers and minor workers. The proportion of soldiers and minor workers in a colony has a major impact on the colony's fitness and is tightly regulated. Here, we summarize over 100 years of research on the internal, external, and developmental factors that regulate subcaste production as well as influence subcaste evolution in Pheidole. We hope that summarizing these factors into a network of interactions will provide insight into how complex biological systems regulate, develop, and evolve.

Comparative Transcriptomics of Alternative Developmental Phenotypes in a Marine Gastropod.

Alternative phenotypes are discrete phenotypic differences that develop in response to both genetic and environmental cues. Nutritive embryos, which arrest their development to serve as nutrition for their viable siblings, are an example of an alternative developmental phenotype found in many animal groups. Females of the marine snail, Crepidula navicella, produce broods that consist mainly of nutritive embryos and a small number of viable embryos. In order to better understand the genetic mechanisms that lead to the development of alternative phenotypes in this species, we compared the transcriptomes of viable and nutritive embryos at the earliest stage that we were able to distinguish visually between the two. Using high-throughput Illumina sequencing, we assembled and annotated a de novo transcriptome and compared transcript levels in viable and nutritive embryos. Viable embryos express high levels of transcripts associated with known developmental events, while nutritive embryos express high levels of apoptosis-related transcripts. Gene Ontology term enrichment with GOSeq found that these are associated with the negative regulation of apoptotic processes. This enrichment, combined with morphological evidence, suggests that apoptosis is important in the formation of gastrula-like nutritive embryos. Apoptosis has been implicated in the development of alternative phenotypes in other animal groups, raising the possibility that this mechanism's role in alternative phenotypes is conserved in gastropod development. We suggest possible alternative mechanisms of nutritive embryo development. Most importantly, we contribute further evidence to the hypothesis that nutritive embryos are an alternative developmental phenotype.

Modeling evolutionary transitions in social insects.

Mathematical models based on direct fitness calculations may be able to explain important aspects of social evolution in insects.

Past climate change on Sky Islands drives novelty in a core developmental gene network and its phenotype.

BACKGROUND: A fundamental and enduring problem in evolutionary biology is to understand how populations differentiate in the wild, yet little is known about what role organismal development plays in this process. Organismal development integrates environmental inputs with the action of gene regulatory networks to generate the phenotype. Core developmental gene networks have been highly conserved for millions of years across all animals, and therefore, organismal development may bias variation available for selection to work on. Biased variation may facilitate repeatable phenotypic responses when exposed to similar environmental inputs and ecological changes. To gain a more complete understanding of population differentiation in the wild, we integrated evolutionary developmental biology with population genetics, morphology, paleoecology and ecology. This integration was made possible by studying how populations of the ant species Monomorium emersoni respond to climatic and ecological changes across five 'Sky Islands' in Arizona, which are mountain ranges separated by vast 'seas' of desert. Sky Islands represent a replicated natural experiment allowing us to determine how repeatable is the response of M. emersoni populations to climate and ecological changes at the phenotypic, developmental, and gene network levels. RESULTS: We show that a core developmental gene network and its phenotype has kept pace with ecological and climate change on each Sky Island over the last ~90,000 years before present (BP). This response has produced two types of evolutionary change within an ant species: one type is unpredictable and contingent on the pattern of isolation of Sky lsland populations by climate warming, resulting in slight changes in gene expression, organ growth, and morphology. The other type is predictable and deterministic, resulting in the repeated evolution of a novel wingless queen phenotype and its underlying gene network in response to habitat changes induced by climate warming. CONCLUSION: Our findings reveal dynamics of developmental gene network evolution in wild populations. This holds important implications: (1) for understanding how phenotypic novelty is generated in the wild; (2) for providing a possible bridge between micro- and macroevolution; and (3) for understanding how development mediates the response of organisms to past, and potentially, future climate change.

The significance and scope of evolutionary developmental biology: a vision for the 21st century.

Evolutionary developmental biology (evo-devo) has undergone dramatic transformations since its emergence as a distinct discipline. This paper aims to highlight the scope, power, and future promise of evo-devo to transform and unify diverse aspects of biology. We articulate key questions at the core of eleven biological disciplines-from Evolution, Development, Paleontology, and Neurobiology to Cellular and Molecular Biology, Quantitative Genetics, Human Diseases, Ecology, Agriculture and Science Education, and lastly, Evolutionary Developmental Biology itself-and discuss why evo-devo is uniquely situated to substantially improve our ability to find meaningful answers to these fundamental questions. We posit that the tools, concepts, and ways of thinking developed by evo-devo have profound potential to advance, integrate, and unify biological sciences as well as inform policy decisions and illuminate science education. We look to the next generation of evolutionary developmental biologists to help shape this process as we confront the scientific challenges of the 21st century.

Epigenetic variation in the Egfr gene generates quantitative variation in a complex trait in ants.

Complex quantitative traits, like size and behaviour, are a pervasive feature of natural populations. Quantitative trait variation is the product of both genetic and environmental factors, yet little is known about the mechanisms through which their interaction generates this variation. Epigenetic processes, such as DNA methylation, can mediate gene-by-environment interactions during development to generate discrete phenotypic variation. We therefore investigated the developmental role of DNA methylation in generating continuous size variation of workers in an ant colony, a key trait associated with division of labour. Here we show that, in the carpenter ant Camponotus floridanus, global (genome-wide) DNA methylation indirectly regulates quantitative methylation of the conserved cell-signalling gene Epidermal growth factor receptor to generate continuous size variation of workers. DNA methylation can therefore generate quantitative variation in a complex trait by quantitatively regulating the transcription of a gene. This mechanism, alongside genetic variation, may determine the phenotypic possibilities of loci for generating quantitative trait variation in natural populations.